1(1 


THE  EXAMINATION  OF  MILK 

FOR 

PUBLIC  HEALTH  PURPOSES 


JOSEPH  RACE,  F.I.C. 

City  Bacteriologist  and  Food  Examiner,  Ottawa;  Chairman  of  Committee  on 

Standard  Methods  of  Analysis,  Canadian  Public  Health  Association, 

Member  of  Committee  on  Municipal  Food  Administration, 

American  Public  Health  Association 


FIRST   EDITION 


NEW  YORK 

JOHN  WILEY  &   SONS,   INC. 

LONDON:  CHAPMAN  &   HALL,   LIMITED 

1918 


Copyright,  1917 

BY 

JOSEPH  RACE 


PRESS   OF 

BRAUNWORTH   *  CO. 

OOOK  MANUFACTURERS 

BROOKLYN     N.   V. 


PREFACE 


THIS  volume  is  primarily  intended  as  a  practical  handbook 
for  those  engaged  in  the  chemical  and  bacteriological  exami- 
nation of  milk  for  public  health  purposes,  but  it  is  also  hoped 
that  it  will  be  of  material  assistance  to  students  and  others  who 
have  previously  assimilated  the  fundamentals  of  bacteriologi- 
cal technique. 

The  control  of  milk  supplies  was  formerly  confined  to  a 
chemical  examination  for  adulteration,  but  since  the  beginning 
of  the  20th  century  the  bacteriological  examination  has  been 
regarded  as  a  "  sine  qua  non,"  and  in  America  the  present 
tendency  is  to  have  both  examinations  made  under  the  super- 
vision of  the  Public  Health  Authorities.  For  this  reason  no 
apology  is  necessary  for  the  inclusion  of  chemical  methods  and 
the  data  which  will  enable  the  examiner  to  interpret  the  results 
obtained. 

In  the  bacteriological  section  an  attempt  has  been  made  to 
include  all  methods  that  have  been  proved  to  be  reliable  and  in 
some  instances  the  details  of  the  standard  methods  of  the 
American  Public  Health  Association  have  been  given;  in  other 
cases  the  report  as  published  by  the  A.P.H.A.  should  be  con- 
sulted. 

The  tables  of  bacteriological  results  have  been  added  in 
the  hope  that  they  will  lead  to  the  standardisation  of  records. 
At  present  the  results  reported  by  many  laboratories  are  not 
comparable  because  of  the  form  in  which  they  are  issued. 

JOSEPH  RACE. 
OTTAWA,  ONT., 
December,  1917. 

415024 


CONTENTS 


CHAPTER  PAGE 

I.  CONSTITUENTS  OP  MILK 1 

Fat.  Lactose.  Proteids.  Salts.  Gases.  Enzymes.  Immune 
bodies.  Physical  constants. 

II.  NORMAL  COMPOSITION  OF  MILK 34 

Average  composition.  Influence  of  brood,  food,  season, 
milking  interval,  and  stage  of  lactation  on  milk  constituents. 
Colostrum.  Abnormal  milk.  Influence  of  disease.  Milk 
adulteration.  Milk  standards. 

III.  CHEMICAL  EXAMINATION 66 

Fat.  Total  Solids.  Ash.  Specific  Gravity.  Solids  Not- 
fat.  Lactose.  Proteids.  Acidity.  Aldehyde  value.  Min- 
eral constituents.  Refraction  of  serum.  Preservatives. 
Coloring  matter.  Milk  products.  Cream.  Enzymes. 

IV.  BACTERIA  IN  MILK 93 

Intra-mammary  milk.  Efforts  to  obtain  sterile  milk.  Fore 
milk  and  stoppings.  Influence  of  washing,  brushing,  dust, 
food,  vessels,  coolers,  and  storage  conditions.  Germicidal 
action.  Development  of  various  organisms  in  milk. 

V.  THE  ENUMERATION  OF  BACTERIA  IN  MILK 113 

Reasons  for  determination  of  total  count.  Relation  of  count 
to  toxicity.  Plating  methods.  Gelatine.  Agar.  Compari- 
son of  media.  Acidity.  Accuracy  of  counts.  American 
standard  method.  Direct  methods  of  Slack,  Stewart,  and 
Breed.  Indirect  methods.  Methylene  blue  test.  Acidity. 

VI.  EXCREMENTAL  ORGANISMS 135 

B.  coli.  Occurrence  of  B.  coli  in  milk.  Estimation  of  B.  coli. 
Enrichment  methods.  Plate  methods.  Classification  of 
type.  B.  enteritidis  sporogenes.  Streptococci. 

VII.  PATHOGENIC  ORGANISMS 150 

Streptococci.  Septic  sore  throat.  B.  diphtherias.  Diphther- 
oid  bacilli.  B.  typhosus.  Gaertner  group.  Morgan's  Ba- 
cillus No.  I.  B.  tuberculosis.  Pseudo  tuberculosis. 


vi  CONTENTS 

CHAPTER  PAGE 

VIII.  CELLS,  DIRT  AND  DEBRIS 171 

Cells.  Epithelial  cells.  Blood  cells.  Estimation  of  cells. 
Centrifugal  methods.  Direct  methods.  Significance. 
Standards.  Dirt  and  Debris.  Nature  of.  Sedimentation 
and  centrifugal  methods.  Filtration  methods.  Significance 
of  dirt. 

IX.  MISCELLANEOUS 185 

Pasteurised  and  Heated  Milk.  Effect  of  heat  on  cream  line, 
peroxidases,  reductase,  albumin,  and  rennin  coagulation. 
B.  abortus.  Acid  producing  organisms.  Aciduric  bacilli. 
Fermentation  test.  Collection  of  samples.  Recording 
results. 

APPENDIX 207 

Composition  of  special  media.     Useful  tables. 

NAME  INDEX 217 

SUBJECT  INDEX 221 


EXAMINATION  OF  MILK 
FOR  PUBLIC  HEALTH  PURPOSES 


CHAPTER  I 
CONSTITUENTS   OF   MILK 

MILK  is  the  opaque  white  fluid  which  is  secreted  by  the 
mammary  glands.  It  consists  essentially  of  an  emulsion  of 
fat  and  a  colloidal  solution  of  caseinogen  in  water  containing 
lactose  and  traces  of  mineral  matter. 

Milk  fat,  with  which  is  associated  small  quantities  of 
cholesterol,  lecithin,  and  a  trace  of  colouring  matter,  consists 
of  a  mixture  of  triglycerides  of  various  fatty  acids.  These 
acids  are  mixtures  of  the  straight  chain  series  CnH2n+iCOOH 
and  CnH2n-iCOOH,  the  less  saturated  acids  being,  accord- 
ing to  the  best  information,  entirely  absent.  The  relative 
proportions  of  the  various  acids  are  by  no  means  constant,  being 
dependent  upon  various  factors  such  as  foodstuffs,  seasonal 
variations,  breed  of  cattle,  and  climatic  conditions. 

The  fat  is  present  in  milk  as  enormous  numbers  of  very 
small  globules  and  it  is  the  reflection  of  light  from  these  par- 
ticles and  those  of  caseinogen  that  produces  the  character- 
istic white  opaque  appearance  of  milk.  Although  it  was  for- 
merly held  that  the  fat  globules  were  surrounded  by  albuminous 
membranes  which  preserved  the  form,  it  is  now  generally 
accepted  that  this  is  due  to  surface  tension  and  that  the  size 
of  the  globules  can  be  altered  by  physical  methods. 

The  size  of  the  fat  globules  in  milk  varies  from  0.8/i  to  20/i 
with  an  average  of  about  2.7/z  and  the  number  of  globules 
from  19X108  to  60X108  per  cubic  centimeter.  Although  no 


2         ;Vc  i    /{«£{{]  iCONSTHOJENTS  OF  MILK 

definite  relation  has  been  established  between  the  breed  of 
cattle  and  the  size  and  number  of  globules  there  are  a  number 
of  results  which  indicate  that  during  interrupted  milking  the 
size  of  the  globules  increases  with  the  fat  content  and  also  that 
as  the  lactation  period  proceeds  the  globules  decrease  in  size 
and  increase  in  number  (see  p.  43). 

The  origin  and  method  of  formation  of  milk  fat  have  not 
been  discovered  although  many  hypotheses  have  been  proposed. 
The  normal  process  seems  to  be  the  formation  of  milk  fat, 
directly  or  indirectly,  from  nutritive  fat,  but  when  this  source 
is  eliminated  the  formation  of  milk  fat  proceeds,  though  dimin- 
ished in  activity,  by  drawing  upon  the  body  fat.  Even  when 
the  body  fat  is  exhausted,  milk  fat  can  be  formed:  this  is  attrib- 
uted to  proteids  acting  as  the  source  of  fat. 

The  various  analytical  and  physical  constants  of  milk  fat 
are: 

0*7  o 

Specific  gravity  -^z 0.9094-0.9140 

o7.o 

Refractive  index,  35°  C 1 . 4550-1 . 4586 

Melting-point 28°  C.-360  C. 

Solidifying  point. 21°  C.-270  C. 

Reichert-Wollny  value 25-27 

Iodine  absorption 31-35 

The  calorific  value  of  butter  fat,  according  to  Stohmann, 
is  9.231  calories  per  gram  and  according  to  At  water,  from  9.320 
to  9.362  calories.  A  value  of  9.3  is  usually  employed  in  cal- 
culating the  calorific  value  of  milk  fat.  The  molecular  weight 
of  fat,  as  calculated  from  the  amount  of  alkali  required  for 
saponification  and  assuming  that  all  the  acids  present  are  mono- 
basic, is  from  720-740,  whilst  the  direct  determination  by  the 
cryoscopic  method  points  to  values  from  696-716.  The 
presence  of  dibasic  acids  would  harmonise  these  two  sets  of 
results,  but  such  acids  have  not  been  isolated  from  butter  fat. 

Lactose.  Although  there  is  some  evidence  of  the  presence 
of  traces  of  a  monosaccharide  in  milk,  the  carbohydrate  secreted 


LACTOSE  3 

under  normal  conditions  is  lactose  or  milk  sugar.  Lactose  is  a 
disaccharide  of  the  empirical  formula  Ci2H22On  and  is  found 
in  the  milk  of  most  mammals.  Lactose  is  secreted  in  the  gland 
and  is  found  only  in  the  milk,  though,  if  suckling  is  interrupted, 
it  may  appear  in  the  urine,  from  which  is  it  eliminated  on  re- 
moval of  the  lactating  gland:  if  the  gland  is  removed  before 
the  lactation  period  commences  it  may  not  appear  at  all.  The 
fact  that  the  blood  in  the  mammary  vein  before  parturition  and 
during  lactation  contains  less  dextrose  than  the  blood  of  the 
jugular  vein  (Kaufman  and  Lagne)  suggests  either  dextrose,  or 
the  constituents  from  which  dextrose  is  formed,  as  the  source 
of  lactose. 

Two  forms  of  lactose  exist  and  are  known  as  the  alpha  and 
beta  varieties.  When  lactose  is  obtained  by  crystallisation 
from  water,  the  alpha  modification,  which  crystallises  in  the 
rhombic  form,  is  formed:  this  modification  exhibits  the  phe- 
nomenon of  multirotation,  i.e.,  shows  a  decreasing  specific 
rotation  with  lapse  of  time  after  solution  in  water.  For  a 
short  period  of  time,  the  length  of  which  depends  upon  the 
temperature,  the  solution  of  alpha  lactose  shows  a  specific 
rotation  of  [a]D=+84.0,  but  this  gradually  diminishes  until  a 
value  of  +52.5  is  reached,  this  being  the  specific  rotation  of  the 
stable  variety  of  lactose  containing  one  molecule  of  water. 
The  corresponding  value  of  the  anhydrous  lactose  is  +55.3. 
Anhydrous  lactose,  obtained  by  heating  the  hydrated  carbo- 
hydrate to  130°  C.,  does  not  produce  multi-rotation  in  aqueous 
solutions.  The  beta  modification,  produced  by  rapid  evapora- 
tion of  aqueous  solutions  of  lactose  in  metal  vessels,  has  a 
specific  rotation  [a]r>+32.7  and  shows  the  same  birotation 

initial  rotation 

ratio,  i.e.,  -^ — r~    — : —  as  the  alpha  modification,  viz.,  1.6. 
final  rotation 


This  shows  that  the  reaction  is  mono-molecular  in  character. 
The  density  of  the  alpha  variety  is  1.545  ^  and  that  of  a 
solution  containing  10  grams  per  100  c.cms./ 1.0391  jf^.  The 
specific  rotation  is  [a]D  =  52.5  at  20°  C.  and  is  lowered  6.075  for 
each  degree  rise  in  temperature.  The  refractive  index  /i£>20°  of 


4  CONSTITUENTS  OF  MILK 

a  solution  containing  10  grams  per  100  c.cms.  is  1.3461  and  of  a 

5  per  cent  solution  1.3395. 

Lactose  is  not  fermented  by  ordinary  yeast  (Saccharo- 
mycetes  cerevicige)  and  is  not  affected  by  the  ordinary  enzymes. 
The  enzyme  lactase,  which  is  capable  of  hydrolysing  lactose 
into  dextrose  and  galactose,  is  found  as  an  endo  enzyme  in 
Torula  kefyr  and  T.  tyrcola  and  also  as  an  exo  enzyme  in 
Kefyr  grains. 


Lactose  Dextrose  Galactose 

Lactase  is  also  widely  distributed  in  the  animal  kingdom, 
being  present  in  the  mucous  membrane  of  the  stomachs  of 
infants  and  also  in  the  expressed  juices  of  muscle,  liver,  lungs, 
and  pancreas.  • 

The  action  of  acids  generally  is  similar  to  that  of  lactase, 
though  the  mineral  acids  are  much  more  effective  than  those 
of  the  organic  series.  Dextrose  and  galactose,  according  to 
Fischer,  have  the  following  constitutional  formulae: 

COH  COH 

I  I 

H— C— OH  H— C— OH 

I  I 

HO— C— H  HO— C— H 

H— C— OH  HO— C— H 

—OH  H— C— OH 

!  I 

CH2OH  CH2OH 

Dextrose  Galactose 

These  formulae  show  both  sugars  to  be  isomeric  aldoses  of 
the  monose  type.  Their  specific  rotatory  powers  [a]D  are 


Dextrose. 

Galactose. 

Equilibrium  form.  . 

52  7 

80  3 

Alpha  modification  

105. 

120 

Birotation  ratio     ...    . 

2 

1  5 

LACTOSE  5 

The  most  important  products  derived  from  lactose,  in  connec- 
tion with  the  bacteriological  examination  of  milk,  are  the  lactic 
acids.  Lactic  acid  (C3HoO3)  exists  as  four  different  isomers, 
three  having  the  constitutional  formula  CH3-CH(OH)-COOH 
or  alpha  hydroxy  propionic  acid,  and  one  CH2(OH)  •  CH2  •  COOH 
hydracrylic  acid  or  beta  hydroxy  propionic  acid.  As  the  latter 
is  not  produced  during  the  bacterial  decomposition  of  lactose 
no  further  description  of  this  acid  is  necessary  in  this  work. 
Alpha  hydroxy  propionic  acid,  or  lactic  acid  as  it  is  usually 
known  as,  contains  an  asymmetric  carbon  atom 

H 

CH3— C— COOH 
OH 

and  exists,  therefore,  in  three  different  forms,  viz.,  dextro, 
Isevo,  and  racemic  or  inactive  lactic  acids.  .The  dextro  and 
Isevo  rotatory  acids  are  both  produced  by  micro-organisms,  but 
unless  pure  cultures  are  employed  the  majority  of  the  acid 
produced  is  of  the  racemic  (d+l)  variety. 

The  density  of  lactic  acid  is  1.2485  ^fs  and  the  refractive 
index  ^20°  1.4469.  On  evaporation  of  aqueous  solutions  of 
lactic  acid  dehydrolactic  acid  CeHioOs  is  produced,  and,  ulti- 
mately, at  higher  temperatures,  lactide  C6HgO4  is  formed.  The 
boiling  point  of  lactic  acid  is  83°  C.  at  1  mm.  pressure  and  119° 
C.  at  12  mm.  pressure.  Lactic  acid,  though  insoluble  in  petro- 
leum ether,  is  soluble  in,  and  miscible  with  alcohol  and  ether 
in  all  proportions. 

Lactic  acid  forms  well-defined  salts  with  various  metals 
and  these  may  be  used  for  the  separation  of  the  acid.  The 
calcium  salt  which  crystallises  with  5  molecules  of  water  is 
soluble  to  the  extent  of  9.5  per  cent  in  cold  water:  zinc  lactate 
(ZnC6HioO4-3H2O)  is  less  soluble,  1.3  per  cent  in  cold  water 
and  13  per  cent  in  hot,  and  forms  well-defined  monoclinic 
prisms. 


6  CONSTITUENTS  OF  MILK 

Proteids.     The  proteids  of  milk  are: 

Per  Cent. 

Caseinogen : approximately  2.0-3.0 

Lactalbumin * . .  approximately  0.3-0.8 

Lactoglobulin a  trace 

Mucoid  proteid a  trace 

Caseinogen*  is  a  distinctly  acid  phospho  proteid  which  does 
not  contain  purine  or  pyrimidine  derivatives.  Lactalbumin,  as 
its  name  implies,  is  one  of  the  albumins  and,  therefore,  soluble 
in  water  and  coagulated  by  heat.  Lactoglobulin  is  insoluble 
in  water  but  soluble  in  salt  solutions. 

According  to  Richmond  the  proteids  of  milk  are  characterised 
by  the  following  reactions :  Caseinogen  is  precipitated  by  adding 
sodium  chloride,  magnesium  sulphate;  or  ammonium  sulphate 
to  saturation:  globulin  is  soluble  in  a  saturated  solution  of 
sodium  chloride  but  is  precipitated  by  magnesium  and  ammo- 
nium sulphates:  albumin  is  soluble  in  saturated  solutions  of 
sodium  chloride  and  magnesium  sulphate  but  is  precipitated 
by  ammonium  sulphate.  Albumin,  however,  may  be  precip- 
itated by  magnesium  sulphate  in  slightly  acid  solutions  but  is 
redissolved  on  neutralisation  of  the  solution.  These  reactions 
are  relatiy.e.jnather  than  specific  and  cannot  be  relied  upon  for 
quantitative  separation  oTTEe" various  proteids:  they  may, 
however,  te  used  for  preparing  the  pure  proteids  by  redissolving 
and  reprecipitating  the  various  fractions.  Other  methods  may 
also  be  used  for  the  separation  of  the  proteids.  For  example, 
the  caseinogen  may  be  removed  by  the  action  of  chymase,  the 
lab  ferment  of  rennet,  or  by  filtration  through  coarse  porcelain : 
filtration  through  fine  porcelain  or  boiling  with  a  small  quan- 
tity of  acid  followed  by  filtration  will  remove  all  the  proteids. 
Lactalbumin  is  slowly  coagulated  by  heating  at  70°  C.,  but 
very  little  is  precipitated  when  the  acidity  is  normal.  Casein- 

*  Caseinogen  is  used  in  these  pages  to  designate  the  mother  substance 
and  paracasein  the  rennet  transformation  product:  this  nomenclature, 
though  not  strictly  logical,  eliminates  the  ambiguity  that  arises  from  the 
difference  in  the  prevailing  English  and  American  phraseology. 


CASEINOGEN  7 

ogen  and  albumin  may  also  be  precipitated  by  the  addition  of  a 
solution  of  calcium  chloride  if  the  milk  is  previously  heated  to 
35°  to  45°  C.  All  three  proteids  are  soluble  in  alkalies  and 
insoluble  in  alcohol  and  ether :  their  copper,  mercuryv  and  other 
salts  of  the  heavy  metals  are  insoluble,  and  all  the  lacto  proteids 
are  completely  precipitated  by  tannin  and  phosphotungstic  acids. 

Caseinogen,  when  pure,  is  a  white,  amorphous,  odourless, 
and  tasteless  substance  which  is  practically  insoluble  in  water. 
The  specific  gravity  is  1.257.  Owing  to  the  stability  of  the 
additive  compound  which  calcium  caseinogenate  forms  with 
calcium  phosphate,  in  which  form  it  is  present  in  milk,  the 
preparation  of  pure  caseinogen  is  a  matter  of  considerable  dif- 
ficulty, and  it  is  probable  that  at  least  a  portion  of  the  differ- 
ences in  composition  found  by  various  observers  is  due  to  this 
factor.  Repeated  precipitation  and  solution  remove  the 
greater  part  of  the  calcium  but  the  last  traces  are  extremely 
difficult  to  eliminate  (Van  Slyke  and  Bosworth1).  Caseinogen 
is  easily  precipitated  by  the  addition  of  a  few  drops  of  glacial 
acetic  acid  to  milk  diluted  with  an  equal  volume  of  water,  and 
the  precipitate  may  be  redissolved  by  the  addition  of  caustic 
alkalies,  alkaline  earths,  ammonia,  carbonates,  bicarbonates,  or 
phosphates,  even  in  minute  quantities.  Schryver2  has  shown 
that  if  the  caseinogen  produced  by  precipitation  with  acetic 
acid  is  allowed  to  remain  in  contact  with  the  excess  of  acid 
(1  in  1000)  at  room  temperature,  or  is  heated  with  wa.ter  to 
37°  C.,  a  product  is  formed  the  solubility  of  which  in  lime  water 
is  only  about  one-third  that  of  natural  caseinogen.  This  has 
been  designated  as  "metacaseinogen,"  the  solution  of  which  in 
half  saturated  lime  water  is  opalescent  but  not  opaque.  Meta- 
caseinogen can  be  reconverted  into  caseinogen  by  solution  in 
sodium  hydrate  and  precipitation  with  acetic  acid  providing 
that  the  contact  with  the  acid  is  not  unduly  prolonged.  Meta- 
caseinogen is  identical  in  composition  with  caseinogen:  the 
following  are  some  of  the  more  authentic  analyses  of  caseinogen. 

Most  of  the  analyses  given  were  obtained  from  material 
prepared  by  Hammerstein's  method,  i.e.,  by  repeated  precip- 


CONSTITUENTS  OF  MILK 
TABLE  I 


C 

H 

0 

N 

S 

P 

Hammerstein  (1883-1885) 

52.96 
53.30 
54.00 
53.07 

7.05 
7.07 
7.04 
7.13 

22.73 
22.03 

21.74 

15.65 
15.91 
15.60 
15.64 
15.45 
15.64 
15.65 
15.67 
15.63 

0.76 
0.82 
0.77 
0.76 
0.76 
0.72 
0.83 
0.77 
1.015 

0.85 
0.87 
0.85 
0.80 
0.77 
0.81 
0.88 
0.82 
0.73 

Chittenden  and  Painter  (1887) 

Lehmann  and  Hempel  (1894)  

Ellenberger  (1902) 

Lacqueur  and  Sackur  (1903)  

Burow  (1905)                               

52.82 
52.69 
53.17 
53.20 

7.09 
6.81 
7.09 
7.09 

22.92 
23.14 

22.48 
22.34 

Tangl  (1908) 

Van  Slyke  and  Bosworth  (1913)  mean  . 
Geake  (1913)                                 .    . 

itation  with  acid  and  solution  in  alkali,  and  it  is  possible  that 
during  this  process  a  portion  of  the  sulphur  was  removed  as 
sulphides  as  the  sulphur  portion  of  the  molecule  is  slightly  un- 
stable. Lehmann's  material  was  obtained  by  filtration  through 
porous  plates  and  probably  contained  a  portion  of  the  lime  salts 
which  constitute  part  of  the  caseinogen  complex  in  milk.  From 
the  percentage  composition,  Richmond  has  calculated  the  em- 
pirical formula  for  caseinogen  to  be  Ci62H25sN4iSPO52,  and  in 
support  of  this  he  quotes  experiments  3  in  which  he  found  that 

N 
•rrr  potassium  and  sodium  carbonate  solutions,  when  treated 

AUU 

with  an  excess  of  caseinogen,  dissolved  1.83  and  1.86  parts  per 
100  c.cms.,  respectively.  The  above  formula,  according  to  Rich- 
mond, would  give  1.84  parts  per  100  c.cms.  The  author  in 
some  unpublished  experiments,  determined  the  solubility  of 

N 
caseinogen  in  -—  KOH  and  obtained  a  value  of  1.83  grams  per 

J.UU 

100  c.cms.  at  room  temperature  (67°  F.) :  other  temperatures, 
however,  gave  different  values,  so  that  these  results  cannot  be 
regarded  as  having  any  bearing  on  the  constitution  or  weight 
of  the  molecule.  Various  compounds  of  caseinogen  with  bases 
have  been  reported.  Soldner4  separated  compounds  of  casein- 
ogen and  lime  containing  1.11  and  1.67  per  cent  of  Ca.,  re- 


CASEINOGEN 


9 


spectively.  Lehmann's  material  as  separated  by  filtration 
contained  1.02  to  1.25  per  cent  of  Ca.  Van  Slyke  and  Bos- 
worth  1  report  four  compounds  with  lime,  containing  0.22, 0.44, 
1.07,  and  1.78  per  cent  of  Ca.  They  also  prepared  compounds 
with  ammonia,  sodium,  and  potassium,  containing  0.20  per 
cent  NH4,  0.26  per  cent  Na,  and  0.44  per  cent  K. 

The  acidity  of  caseinogen  has  been  determined  by  many 
observers  with  fairly  good  agreement.  The  more  important 
results  are: 


1  c.c.  —  NaOH  equals 

1  gram  Caseinogen 
equals 

Lacqueur  and  Sackur  
IVlathaiopoulos 

0.1138  gr.  caseinogen 
0  11315 

8.81  c.c.  ^NaOH 
8.84 

Loner 

0.1124 

8.90 

Van  Slyke  and  Bosworth  .  .  . 

0.1111 

9.00 

From  the  analysis  of  the  lime  salts,  Van  Slyke  and  Bosworth 
regard  caseinogen  as  an  octobasic  acid  and  classify  these  salts  as 
follows : 


GRAMS  PEB  100 

GRAMS 

REACTION  TO 

CASEINOGEN. 

Name  of  Compound. 

Valencies 
Satisfied 

Phenol  ' 

Ca 

CaO 

Litmus. 

Phthalein. 

0  22 

0  31 

Monocalcium  caseinogenate 

1 

044 

0  62 

Di  calcium  caseinogenate 

2 

1.07 

1.50 

Neutral  calcium  caseinogenate 

Neutral 

Acid 

5 

1.78 

2.50 

Basic  calcium  caseinogenate 

Neutral 

8 

From  a  consideration  of  the  dissociation  values  of  caseino- 
genates  in  dilute  solutions,  Lacqueur  and  Sackur5  regarded 
caseinogen  as  either  a  penta  or  hexabasic  acid  but  a  later  inves- 
tigation of  the  physical  properties  by  Robertson6  shows  that  it  is 


10  CONSTITUENTS  OF  MILK 

octobasic.  This  would  give  a  molecular  weight  of  approxi- 
mately 8900. 

Caseinogen,  when  dissolved  in  dilute  alkali,  has  a  pronounced 
laevo  rotatory  action  on  polarized  light,  but  the  specific  rota- 
tion is  not  constant,  varying  from  —94.8  to  —111.8,  according 
to  the  concentration  and  nature  of  the  alkali  used  as  the  solvent 
(Long).  The  soluble  salts  of  caseinogen  may  be  divided  into 
two  classes  (1)  salts  of  the  alkaline  earths,  and  (2)  salts  of  the 
alkalies.  According  to  Osborne7  these  are  distinguished  by 
the  inability  of  the  former  to  pass  through  the  film  of  the 
Martin  gelatin  filter  and  by  the  formation  of  opalescent  solu- 
tions. The  solutions  of  the  second  class  filter  through  gelatin 
membranes  and  are  translucent.  Both  classes  of  salts  are 
neutral  to  phenolphthalein  when  the  valency  of  caseinogen  is 
entirely  satisfied,  but  when  litmus  is  used  as  the  indicator  no 
definite  change  is  indicated  and  the  point  of  neutralisation 
varies  with  the  concentration  of  the  solution  (Schryver). 
Salts  of  copper,  mercury,  and  lead,  precipitate  caseinogen 
from  neutral  solutions,  and  mercury  salts  are  also  effective  in  the 
presence  of  acid:  the  precipitates  so  obtained  are  not  constant 
in  composition  but  vary  with  the  conditions  obtaining.  The 
insolubility  of  the  compounds  with  the  heavy  metals  is  utilised 
in  milk  analysis  in  the  preparation  of  protein  free  milk  serum 
for  use  in  the  polarimeter  and  refractometer.  Caseinogen  also 
exhibits  basic  properties  and  combines  with  acids  with  the 
formation  of  clear  solutions.  Long8  found  that  1  gram  of 

N 
caseinogen  combined  with  about  7  c.cms.  of  —  acid  in  the  form 

of  sulphuric,  hydrochloric,  hydrobromic,  hydriodic,  and  acetic 
acids  to  form  soluble  salt  like  compounds.  Some  observers 
have  stated  that  precipitated  caseinogen  also  combined  with 
acids  but  L.  L.  Van  Slyke  and  D.  D.  Van  Slyke  9  have  shown 
that  the  observed  loss  of  acid  on  precipitation  was  due  to  sur- 
face adsorption  and  depended  upon  the  nature  and  concen- 
tration of  the  acid,  the  temperature,  the  duration  of  contact, 
and  the  degree  of  agitation. 


CASEINOGEN  11 

When  caseinogen  is  acted  upon  by  formaldehyde,  the  amino 
groups  condense  with  the  H-CHO  to  form  methylene  deriva- 
tives. The  resultant  compounds  are  not  digested  by  trypsin 
but  can  be  decomposed  by  steam  and  the  formaldehyde  quan- 
titatively recovered  in  the  distillate.  On  the  formation  of 
methylene  derivatives,  the  alkalinity  due  to  amino  groups  dis- 
appears, and  the  caseinogen  salt,  which  before  condensation 
reacted  neutral  to  phenolphthalein,  becomes  acid  and  can  be 
quantitatively  titrated  with  alkalies.  This  reaction  is  the 
basis  of  the  aldehyde  value  (vide  p.  75). 

Caseinogen,  on  hydrolysis  by  pepsin,  trypsin,  or  dilute 
acids,  undergoes  proteoclastic  digestion  with  the  formation  of 
caseinogen  proteoses  or  caseoses,  as  they  have  been  called,  which 
are  soluble  in  water.  These  caseoses  have  been  subdivided 
into  proto  and  deutero  caseoses  by  their  solubility  in  ammo- 
nium sulphate  solutions  of  certain  concentration. 

The  ultimate  products  of  the  hydrolysis  of  caseinogen  have 
been  extensively  investigated  and  the  results  of  various  ob- 
servers, obtained  with  caseinogen  from  various  sources,  are 
given  in  Table  II. 

Caseinogen  exists  in  milk  as  a  salt  combined  with  phos- 
phate of  calcium,  and  although  the  composition  of  this  com- 
plex has  been  investigated  by  many  chemists  during  the  last 
sixty  years,  it  is  impossible  even  yet  to  state  that  it  is  defi- 
nitely established.  Richmond,  from  an  analysis  of  the  mate- 
rial separated  by  filtration  through  a  porous  cell,  assumes  that 
caseinogen  exists  in  milk  as  a  double  calcium  sodium  caseino- 
genate  combined  with  half  a  molecule  of  tricalcic  phosphate. 
Ci62H255N4iSPO52-Ca-Nai(Ca3P208).  The  quantity  of  acid 
required  for  the  displacement  of  the  sodium  atom  in  this  formula 
by  hydrogen,  would  be  8.3  c.cms.  of  normal  acid  per  litre  of 
milk,  and  Richmond  found  that  on  adding  8.6  c.cms.  N.  hydro- 
chloric acid  or  sulphuric  acid,  the  caseinogen  was  precipitated 
on  boiling,  and  that  the  acidity  of  the  serum  was  equal  to  that 
of  the  milk  after  boiling.  L.  L.  Van  Slyke  and  Bosworth10 
have  pointed  out  that  deductions  based  on  the  acidity  of  milk 


12  CONSTITUENTS  OF  MILK 

TABLE  II 
CASEINOGEN  HYDROLYSIS  PRODUCTS 


Products  of  Hydrolysis. 

Cow's  MILK. 
(Abderhalden,  Fischer, 
Osborne  and  Greed, 
Morner,  Fischer  and 
Abderhalden,  Hart.) 

GOAT'S  MILK. 

(Abderhalden 
and 
Schittenhelm.) 

HUMAN  MILK. 

(Abderhalden 
and 
Schittenhelm.) 

Glycine 

o 

o 

Alanine  

0.90 

1.50 

Valine  

1.00 

Leucine 

10  50 

7.40 

Phenylalanine 

3  20 

2  75 

~; 

Tyrosine  

4.50 

4.95 

4  71 

Serine.    .  .  . 

0.45 

Cystine 

0  06 

Proline  

6.70 

4.60 

Oxyproline  
Aspartic  acid 

1.50 
1  20 

1  20 

Glutamic  acid  
Tryptophane.  . 

11.00 
1.50 

12.00 

Arganine   . 

4  84 

Lysine 

5  80 

Hist  idine  

2.59 

Diaminotrioxy- 
dodecanic  acid  
Ammo  valeric  acid  
Ammonia.                 .    . 

0.75 
7.20 
1  60 

and  milk  serum,  as  determined  in  the  usual  way  by  direct  titra- 
tion  with  alkali,  may  be  entirely  fallacious  because  of  the  errors 
introduced  by  titrating  phosphoric  acid  in  the  presence  of  lime 
salts.  Cameron  and  Hurst11  have  shown  that  the  following 
reactions  may  occur. 

(1)  CaHP04  +2H20       =Ca(OH)2+H3PO4. 

(2)  2CaHPO4+Ca(OH)2  =  Ca3P208  +2H20. 


These  result  in  the  presence  of  free  phosphoric  acid  in  place 
of  neutral  dicalcium  phosphate  and  the  acidity  is,  therefore, 


CASEINOGEN  13 

apparently  higher.  When  milk  is  filtered  through  porcelain, 
the  acidity  of  the  serum  is  usually  approximately  half  that  of 
the  original  milk  on  direct  titration  with  alkali,  but  Van  Slyke 
and  Bosworth  have  shown  that,  if  before  determining  the  acidity, 
the  lime  salts  are  previously  removed  by  precipitation  with 
neutral  potassium  oxalate,  the  acidity  of  the  serum  is  equal  to 
that  of  the  milk :  in  other  words,  the  caseinogen  calcium  phos- 
phate complex  in  milk  is  not  acid  to  phenolphthalein  but  neu- 
tral. Van  Slyke  and  Bosworth 12  filtered  milk  through  porcelain 
but  instead  of  analysing  the  precipitate,  compared  the  serum  and 
the  original  milk.  This  eliminates  errors  caused  by  the  absorp- 
tion of  soluble  salts  if  the  first  filtrates  of  serum  are  rejected. 
Their  results  show  that  caseinogen  exists  in  milk  as  neutral 
calcium  caseinogenate  (caseinogen,  Ca/t)  and  neutral  dicalcium 
phosphate.  These  are  not  in  chemical  combination  as  they 
could  be  almost  completely  separated  by  mechanical  methods. 

The  reaction  of  caseinogen  with  rennin,  a  lab  ferment,  is 
of  considerable  importance  because  of  the  information  it  yields 
regarding  the  constitution  of  caseinogen,  and  also  on  account 
of  the  presence  of  this  ferment  in  the  mucous  lining  of  calves' 
stomachs  and  the  similarity  of  its  action  to  that  of  the  gastric 
juices  of  the  human  stomach.  Although  this  reaction  has  been 
the  subject  of  probably  more  investigations  than  any  other  sub- 
ject in  biological  chemistry  the  modus  operandi  and  the  nature 
of  the  reaction  products  are  still  comparatively  obscure. 

It  has  long  been  known  that  fresh  milk  coagulates  in  the 
stomachs  of  the  higher  animals.  An  aqueous  extract  of  the 
inner  lining  of  the  stomach  of  the  calf  causes  curdling  and  clots 
milk  producing  a  semi-solid  mass.  These  facts  have  been 
utilised  since  an  early  date  in  the  manufacture  of  cheese. 

The  earlier  views  concerning  the  nature  of  this  change  need 
not  be  considered  in  detail  as  they  have  since  been  proved  to 
be  entirely  erroneous.  The  one  most  commonly  accepted 
regarded  the  action  as  one  of  decomposition  of  the  milk  sugar 
into  acids,  which  directly  or  indirectly  produced  the  phe- 
nomenon observed.  The  first  important  advance  was  made 


14  CONSTITUENTS  OF  MILK 

when  Heintz  13  found  that  the  muscosa  extract  of  stomachs 
possessed  the  property  of  clotting  milk  of  an  alkaline  reaction. 
Hammerstein,14  and  Schmidt,15  first  showed  that  the  coagulation 
of  milk  by  rennin  was  due  to  a  soluble  ferment  which  was  named 
"  labferment  "  or  "  chymosin."  Hammerstein  thoroughly  in- 
vestigated the  nature  of  the  reaction  and  his  conclusions  met 
with  fairly  general  acceptance  until  a  few  years  ago.  He 
showed  that  caseinogen  was  not  in  true  solution  in  milk  but  in  a 
state  of  colloidal  suspension,  and  that  the  presence  of  a  certain 
quantity  of  calcium  phosphate  was  necessary  for  the  reaction 
to  occur:  also  that  during  the  reaction  the  caseinogen  was  so 
altered  that  it  was  unable  to  remain  in  colloidal  suspension 
and  was  precipitated  in  the  presence  of  calcium  phosphate  as 
paracasein  calcium  phosphate.  He  further  found  that  the 
caseinogen  was  split  into  at  least  two  other  proteids,  casein  (der 
Kase)  better  described  as  paracasein,  and  whey  proteid  (Mol- 
keneiweiss).  These  were  distinguished  by  the  insolubility  in 
water  of  the  calcium  salts  of  the  former  compared  with  the 
smaller  molecule  of  the  latter  and  the  solubility  of  its  calcium 
salts.  The  composition  of  these  proteins  according  to  Koster 
is  shown  in  Table  III. 

TABLE  III 


Paracasein. 

Whey  Proteid. 

Carbon  

52.88 

50  33 

Hydrogen        .        .    .        

7  00 

7  00 

Nitrogen 

15  84 

13  25 

Phosphorous  (Richmond)  

0.99 

From  these  figures  Richmond  has  calculated  the  approximate 
formulae  for  these  substances  to  be. 


Paracasein 
Whey  proteid 

Hammerstein  concluded  that  the  conversion  of  caseinogen  into 


CASEINOGEN  15 

paracasein  was  independent  of  the  calcium  salts  present  and 
this  has  been  confirmed  by  later  observers.  Some  chemists 
(Loevenhart16  and  Briot17),  have  claimed  that  an  essential  part 
of  the  rennin  reaction  is  a  modification  of  the  mineral  con- 
stituents, but  Harden  and  Macallum18  have  recently  shown  that 
if  caseinogen  solutions  are  treated  with  sufficient  rennin 
(1  :  1000)  no  addition  of  calcium  salts  is  required:  Schryver2 
found  that  clot  formation  could  be  obtained  in  the  entire  absence 
of  calcium  ions.  Duclaux 19  was  the  first  to  find  that  no  proteo- 
clastic  cleavage  is  produced  by  the  action  of  rennin  and  this  has 
been  confirmed  by  Van  Slyke  and  Bosworth,20  Geake,21  and 
Harden  and  Macallum.18  Loevenhart 16  suggested  that  caseino- 
gen and  paracasein  were  chemically  identical  and  that  the  differ- 
ences in  behaviour  were  due  to  changes  in  molecular  association 
or  aggregation.  This  view  is  supported  by  Van  Slyke  and  Hart22 , 
and  Van  Slyke  and  Bosworth  (vide  supra)  who  suggested  that 
calcium  caseinogenate  is  split  by  the  action  of  rennin  into  two 
molecules  of  calcium  paracaseinate  which  is  identical  in  per- 
centage composition  with  the*  original  substance.  Liwschiz23 
attempted  to  differentiate  caseinogen  and  paracasein  by  biolog- 
ical methods.  Three  methods  were  tried,  precipitation,  com- 
plement binding,  and  anaphylaxis,  and  of  these  only  comple- 
ment binding  gave  positive  results  under  certain  conditions. 
The  other  two  methods  entirely  failed  to  distinguish  between 
the  two  substances.  Schryver2  has  suggested  that  all  the 
substances  necessary  for  clot  formation  pre-exist  in  milk  and 
that  aggregation  is  prevented  by  the  absorption  of  simpler 
molecules  from  the  system.  He  formed  the  conception  that  a 
ferment,  for  which  the  colloidal  substances  could  act  as  a  sub- 
strate, could  clear  the  surface  of  such  substances  of  adsorbed 
bodies  and  thus  allow  aggregation  (clot)  formation  to  take 
place.  He  found  that  milk  serum,  Witte's  peptone,  or  glycine, 
inhibited  clot  formation  by  rennin,  and  also  that  apparently 
typical  milk  clots  could  be  formed  by  the  addition  of  calcium 
chloride  to  calcium  caseinogenate  solutions  and  warming. 
These  differ  from  rennin  clots,  however,  in  their  ability  to  pro- 


16  CONSTITUENTS  OF  MILK 

duce  clottable  solutions  on  dispersion  by  acidification  after 
solution  in  alkali.  Clots  produced  by  the  action  of  rennin 
cannot  be  redispersed,  a  fact  that  suggests  some  alteration  in 
structure.  Schryver  found  that  calcium  caseinogenate  solu- 
tions on  warming,  and  sodium  caseinogenate  solutions  after 
treatment  with  carbon  dioxide  in  the  cold,  would  produce  clots 
with  rennin  and  suggested  that  these  observations  point  to  the 
formation  of  caseinogen  by  the  action  of  heat-  in  the  former,  and 
carbon  dioxide  in  the  latter,  and  that  clot  formation  is  produced 
by  the  action  of  rennin  on  the  free  caseinogen  or  metacasein- 
ogen  (see  p.  7). 

Some  observers  have  stated  that  a  change  in  reaction  occurs 
during  the  action  of  rennin  but  Hewarden  24  found  that  hydrogen 
ions  were  not  necessary  for  the  coagulation  of  milk  or  solutions 
of  caseinogen  containing  calcium.  The  author  has  found  that 
the  curd  produced  from  milk  by  rennin  usually  has  an  acidity 
equivalent  to  8.3  to  8.8  c.cms.  of  normal  acid  per  litre  of  milk, 
an  amount  which  is  identical  with  the  acidity  of  the  caseinogen 
in  the  solution  from  which  it  is  produced. 

Caseinogen  is  also  clotted  by  the  action  of  trypsin  and  other 
enzymes,  but  in  the  case  of  trypsin  there  is  definite  evidence  of 
proteoclastic  cleavage  with  the  formation  of  soluble  com- 
pounds containing  nitrogen  and  phosphorous. 

Heating  milk  to  70°  C.  and  upwards,  retards  the  velocity 
of  the  rennin  reaction  by  partial  destruction  of  the  enzyme  and 
precipitation  of  the  calcium  salts:  refrigeration  also  prevents 
the  formation  of  the  characteristic  curd  but  this  property  is 
regained  on  heating  to  37°  C.  (Morgenrath). 

The  optimum  reaction  temperature  for  rennin  is  about  40°  C. 
and  at  temperatures  exceeding  this  it  is  gradually  weakened  and 
finally  destroyed:  the  destruction  by  heat  follows  the  law  of  a 
monomolecular  reaction.  The  velocity  of  the  rennin  reaction 
follows  the  usual  laws  until  40°  C.  is  reached  when  the  observed 
values  become  smaller  than  the  calculated  values  owing  to 
partial  weakening  of  the  enzyme  by  heat.  Some  of  the  results 
obtained  by  Field  on  this  subject  are  given  in  Table  IV. 


LACTALBUMIN 
TABLE  IV 


17 


\ 

T 

K 

K 

Temperature. 

Time  in  Seconds. 

,  10,000 

Calculated. 

25 

54 

185 

185 

30 

32 

312 

327 

35 

17 

588 

574 

40 

10.2 

980 

980 

44 

9 

1111 

1491 

50 

14.7 

680 

2742 

The  time  required  for  the  coagulation  of  milk  by  rennin, 
other  conditions  being  equal,  is  inversely  proportional  to  the 
concentration  of  the  enzyme.  Acids  and  salts  of  the  alkaline 
earths  accelerate  the  reaction,  while  alkalies,  albumoses,  and 
large  amounts  of  neutral  salts,  retard  it:  the  fat  content  also 
influences  the  velocity  of  the  reaction.  The  reaction  can  be 
inhibited  by  the  addition  of  normal  horse  serum  and  a  similar 
effect  is  produced  by  the  anti-rennin  prepared  by  Morgenrath  25 
by  repeated  injection  of  rennin  into  the  blood  stream  of  rabbits. 
As  the  inhibitory  action  of  horse  serum  can  be  prevented  by 
neutralisation  with  acid  (Raudnitz  and  Jakoby)  it  seems  prob- 
able that  both  horse  serum  and  anti-serum  act  by  fixation  of 
the  calcium  ions. 

Lactalbumin.  This  constituent  of  milk  has,  according  to 
Sebelien,  the  following  composition : 


Carbon. 

Hydrogen. 

Nitrogen. 

Sulphur. 

Oxygen. 

Lactalbumin  

52.19 

7.18 

15.77 

1.73 

23.13 

These  results  show  that  the  essential  difference  in  compo- 
sition between  the  albumin  of  milk  and  the  phospho  proteid 
(caseinogen)  lies  in  the  absence  of  phosphorus  in  the  former  and 
its  markedly  higher  content  of  sulphur. 

Lactalbumin  follows  the  general  reactions  of  other  albumins 
in  being  soluble  in  neutral  saturated  solutions  of  magnesium 


18  CONSTITUENTS  OF  MILK 

sulphate  but  is  precipitated  by  the  addition  of  small  quantities 
of  acetic  acid.  It  is  stated  that  lactalbumin  may  be  obtained 
in  a  crystalline  form  by  diluting  the  saturated  magnesium  sul- 
phate solution  with  an  equal  volume  of  water  and  setting  aside 
after  the  addition  of  acetic  acid  until  permanently  turbid. 

Lactalbumin  is  also  precipitated  by  sodium  and  ammonium 
sulphates  when  added  to  saturation.  Tannin,  phosphotungstic 
acid  and  other  general  reagents  also  precipitate  lactalbumin: 
the  salts  of  the  heavy  metals  are  insoluble  in  water.  Lactal- 
bumin is  insoluble  in  alcohol  and  this  reagent  may  be  employed 
for  the  precipitation  of  lactalbumin  from  aqueous  solutions: 
the  precipitate  so  obtained  is  easily  soluble  in  water. 

Lactalbumin  is  a  white  powder  possessing  neither  taste  nor 
odour.  It  coagulates  at  70°  C.  but  the  precipitation  is  never 
complete.  The  specific  rotatory  power,  according  to  Bechamp, 
is[a]z>  =  —  67.5,  but  Sebelein  obtained  values  varying  from 
-36.4  to  -38.0.  Lindet 26  obtained  a  value  of  only  -30.0,  so 
that  apparently  the  preparations  of  both  Bechamp  and  Sebelein 
were  mixtures  of  lactalbumin  with  some  other  substance,  prob- 
ably caseinogen  [«]/>=— 119,  having  a  much  higher  rotatory 
power. 

Lactoglobulin.  Comparatively  little  is  known  regarding 
the  globulin  constituent  of  milk.  It  is  precipitated  by  neutral 
sulphates  such  as  magnesium  sulphate  but  is  quite  soluble  in 
sodium  chloride  solutions  even  after  acidification.  It  is  not 
clotted  by  rennin  but  coagulates  under  the  action  of  heat  alone 
at  a  temperature  of  72°  C.  (Hewlett). 

Probably  not  more  than  0.1  per  cent  of  lactoglobulin  is 
present  in  normal  milk  although  considerably  more  may  be 
found  in  colostrum. 

Mucoid  Proteid.  This  substance,  according  to  Storch, 
contains  14.76  per  cent  of  nitrogen  and  2.2  per  cent  of  sulphur. 
It  is  a  greyish  white  powder  which  is  slightly  soluble  in  dilute 
sodium  and  potassium  hydrates  though  insoluble  in  ammonium 
hydrate,  acetic,  and  hydrochloric  acids.  Mucoid  proteid  gives 
the  usual  proteid  reactions  with  Millon's  reagent  (red),  and 


SALTS 


19 


iodine  (brown),  and  the  xantho  proteic  reaction.  On  hydrolysis 
with  hydrochloric  acid  it  yields  a  quantity  of  a  substance*  capa- 
ble of  reducing  Fehling's  copper  solution. 

This  proteid  is  probably  identical  with  the  /3  casein  of 
Strewe  who  separated  it  from  a  casein  (caseinogen)  by  dissolving 
out  the  latter  with  ammonium  hydrate. 

Salts.  In  addition  to  the  various  acids  and  bases  which 
form  part  of  the  caseinogen  complex,  the  serum  of  milk  contains 
various  salts  in  solution.  The  average  percentage  of  ash  in 
milk  is  about  0.75  per  cent  but  fluctuates  considerably.  The 
average  composition  of  the  ash  of  milk,  as  obtained  by  igni- 
tion is  given  in  Table  V. 

TABLE  V 
COMPOSITION  OF  ASH  OF  MILK  (RICHMOND) 


Per  Cent. 


Lime 20 . 27 

Magnesia 2 . 80 

Potash 28.71 

Soda 6.67 

Phosphoric  acid 29 . 33 

Chlorine .' .  14.00 

Carbon  dioxide 0 . 97 

Sulphuric  acid Trace 

Ferric  oxide 0 . 40 

103.15 
Less  oxygen  =  Cl 3 . 15 

100.00 
Distribution  of  the  phosphoric  acid. 

Grams  per  100  c.cms. 

P2O6  as  caseinogen  combined  with  NaCa 0 . 0605 

P,O5  as  Ca-P2O8 0.0625 

P2O8  as  R3HPO4 0.0770 

P2O5  as  RH2PO4 0.0200 


Total..  .  0.2200 


20 


CONSTITUENTS  OF  MILK 


The  following  results  of  Van  Slyke  and  Bosworth  12  show  the 
composition  of  milk  serum  as  separated  by  filtration  through 
porcelain  candles. 

TABLE  VI 
COMPOSITION  OF  MILK  AND  MILK  SERUM 


Original 
Milk. 

Milk 
Serum. 

Percentage  of 
Milk  Constituents 
in  Serum. 

Sugar.  . 

5.75 

5.75 

100.0 

Caseinogen                              

3  07 

0.00 

Nil 

Albumin 

0  506 

0  188 

37  1 

Nitrogen  in  other  compounds  

0.049 

0.049 

100.0 

Citric  acid. 

0  237 

0  237 

100  0 

Phosphorus  (organic  and  inorganic) 
Phosphorus  (organic)  

0.125 
0  087 

0.067 
0  056 

53.6* 
64  4 

Calcium 

0  144 

0  048 

33  3 

Magnesium  

0.013 

0.007 

53  8 

Potassium  

0  120 

0  124 

100  0 

Sodium 

0  055 

0  057 

100  0 

Chlorine 

0  076 

0  081 

100  0 

Ash.    .    .                   . 

0  725 

0  400 

55  2 

*  Not  obtained  on  same  sample. 

Van  Slyke  and  Bosworth  suggest  that  the  various  combina- 
tions of  acids  and  bases  in  milk  are: 

Proteins  combined  with  calcium  ......................  3  .  20 

Di-calcium  phosphate  (CaHPO4)  .....................  0.  175 

Calcium  chloride  ...................................  0  .  119 

Mono-magnesium  phosphate  (MgH4P2Os)  .............  0.103 

Sodium  citrate  (NasCeHsO?)  ........................  0.222 

Potassium  citrate  (KaCeH^OT)  .......................  0.052 

Di-potassium  phosphate  (K^HPCU)  ...................  0.230 


Other  constituents  which  have  been  found  in  minute  traces 
are  fluorine,  iodine,  silica,  acetates,  and  thiocyanates. 

Lecithin.  C44H9oOgNP  also  exists  in  milk  in  minute  quan- 
tities. 


ENZYMES  21 

Gases.  There  is  no  definite  evidence  of  the  existence  of 
gases  in  milk  as  drawn  from  the  udder,  but,  during  this  process, 
it  absorbs  the  normal  constituents  of  the  air.  Two  analyses  of 
milk  gases  by  Winter  Blyth  are  given  in  Table  VII. 

TABLE  VII 
COMPOSITION  OF  GASES  IN  MILK 


Fresh  Milk. 

Milk  after  Standing 
Two  Hours. 

Carbon  dioxide  

Cubic  centimeters  p« 
0.06 
19.13 
77.60 

T  1000  c.cms.  of  milk 
60.47 
9.30 
30.21 

Oxygen 

Nitrogen  

Blyth  found  that,  on  standing,  the  oxygen  usually  dis- 
appeared in  about  twenty-four  hours  and  that  the  carbon  dioxide 
content  increased  until  it  finally  reached  over  95  per  cent  of  the 
total  gases,  the  residue  being  nitrogen. 

Enzymes.  It  has  been  indubitably  proved  that  fresh  milk 
contains  a  number  of  the  substances  known  as  enzymes,  bodies 
which  are  remarkable  on  account  of  certain  properties  which 
they  possess.  Small  quantities  appear  to  be  capable  of  pro- 
ducing radical  chemical  changes  without  themselves  under- 
going alterations,  although  their  activity  is  diminished  by  the 
transformation  products. 

Enzymes  are  specific  in  character,  i.e.,  only  certain  specific 
enzymes  are  capable  of  acting  upon  certain  compounds,  and 
this  property  has  led  to  the  adoption  of  a  nomenclature  which 
classifies  the  enzyme  in  accordance  with  the  nature  of  the  com- 
pound acted  upon  or  the  nature  of  the  action  produced.  For 
example,  the  enzyme  acting  upon  amylose  is  known  as  amylase, 
whilst  lactase,  glucase,  and  protease,  act  upon  lactose,  glucose, 
and  protein,  respectively:  oxidases  and  reductases  oxidise 
and  reduce,  and  catalase  acts  as  a  catalytic  agent. 

Enzymes  are  thermolabile,  have  optimum  temperatures  of 


22  CONSTITUENTS  OF  MILK 

reaction,  and  are  injuriously  influenced  by  toxins  and  various 
salts.  As  they  have  never  been  isolated  in  a  pure  condition, 
comparatively  little  is  known  as  to  their  composition  and  it  is 
by  their  properties  rather  than  differences  in  composition  that 
enzymes  are  recognised. 

Amongst  the  various  enzymes  that  have  been  discovered 
in  milk  are  amylase,  galactase,  lipase,  lactokinase,  peroxidase, 
reductase,  and  catalase. 

Amylase.  Bechamp  27,  in  1883,  prepared  an  amylase  from 
human  milk  that  converted  soluble  starch  into  sugar  as  readily 
as  amylases  from  other  sources.  The  presence  of  amylase-in 
cows'  milk  has  been  denied  by  Moro,  der  Velde,  Landtsheer, 
and  Kastle  and  affirmed  by  Zaitschick,  Koning,  Seligman, 
Jensen,  and  others.  The  author  has  invariably  found  amylase 
to  be  present,  although  only  in  minute  quantities. 

Galactase.  This  protease  was  first  found  in  mine  by  Bab- 
cock  and  Russell  in  1897  28.  They  found  that  fresh  centrifuge 
slime  showed  proteolytic  properties  even  when  all  bacterial 
activity  was  checked  by  the  presence  of  antiseptics.  Wender  29 
has  shown  that  the  galactase  prepared  from  centrifugal  slimes 
is  not  a  pure  enzyme  but  a  mixture  of  galactase  with  peroxidases 
and  catalase.  The  presence  of  catalase  in  milk  has,  however, 
been  confirmed  by  von  Freudenreich,  Jensen,  Spolverini,  and 
others.  The  action  of  galactase  on  proteids  is  very  similar 
to  that  of  trypsin,  proteoses  and  peptones  being  the  inter- 
mediate, and  amino  acids  the  final  products. 

Lactokinase,  a  kinase  similar  to  enterokinase,  and  a  fibrin 
ferment  have  also  been  found  in  minute  quantities. 

Lipase,  the  enzyme  capable  of  hydrolysing  glycerides  of 
fatty  acids  such  as  monobutyrin,  was  found  in  milk  by  Marfan 
and  Gillet 30.  Cows'  milk  was  found  to  have  a  lipolytic  activity 
of  6-8  on  Hanriot's  scale  as  against  20-30  for  human  milk. 

Salolase.  That  human  and  asses'  milk  have  the  property 
of  hydrolysing  phenyl  salicylate  (salol)  was  observed  by  Nobe*- 
court  and  Merklen.31  The  existence  of  this  ferment  in  milk 
was  disputed  by  Desmouli£rs  and  also  by  Mule  and  Willem, 


CATALASE  23 

who  found  that  the  hydrolysis  was  really  a  saponification 
effected  by  the  presence  of  alkali  and  that  only  alkaline  milks 
showed  the  presence  of  salolase.  Rullman,  in  1910,  proved 
that  milk  obtained  with  aseptic  precautions  did  not  give  the 
salol  splitting  reaction.  It  has  been  suggested  that  salolase  is 
of  bacterial  origin,  although  this  view  is  unsupported  by  experi- 
mental data. 

Peroxidases.  Although  Rullman  has  found  traces  of  sub- 
stances in  milk  capable  of  effecting  oxidation  by  utilisation  of 
atmospheric  oxygen  (true  oxidases),  the  peroxidases  are  much 
more  important.  These  ferments  decompose  hydrogen  perox- 
ide in  accordance  with  the  equation  H202  =  H20+O.  The 
presence  of  nascent  oxygen  is  ascertained  by  the  addition  of 
some  substance  which  undergoes  a  colour  change  on  oxidation 
(a  chromogen).  Benzidine,  guiacol,  ortol,  amidol,  p.  pheny- 
lenediamine,  and  phenolphthalin  have  been  employed  for  this 
purpose.  Kastle  and  Porch32  showed  that  the  power  of  milk 
to  induce  the  oxidation  of  phenolphthalin  and  other  leuco 
bases  by  hydrogen  peroxide  is  greatly  intensified  by  the  addi- 
tion of  certain  substances  of  the  phenol  type. 

Catalase.  Catalase  (Loew)  or  superoxidase  (Raudnitz) 
like  peroxidase  has  the  property  of  decomposing  hydrogen 
peroxide,  but,  instead  of  atomic  oxygen  being  produced  and 
absorbed  by  some  compound  present,  molecular  oxygen  is 
formed  and  may  be  collected  in  the  gaseous  form. 


Some  authors  have  included  catalase  with  the  reductases  in 
accordance  with  the  view  that  the  oxygen  liberated  is  utilised  in 
an  oxidation  process  and  that  the  reaction  is  essentially  one  of 
the  reduction  of  hydrogen  peroxide  to  water.  There  is,  how- 
ever, as  little  basis  for  the  inclusion  of  catalase  with  the  reduc- 
tases as  with  the  peroxidases,  for,  although  its  action  is  inter- 
mediate between  the  two,  it  is  entirely  independent  of  them 
and  well-defined  in  character. 


24  CONSTITUENTS  OF  MILK 

Reductases.  The  ferments  which  cause  the  abstraction  of 
oxygen  from  compounds  without  the  production  of  gaseous 
oxygen,  have  been  termed  reductases.  The  essential  differ- 
ence between  this  reaction  and  that 'produced  by  catalases  is 
in  the  utilisation  or  transference  of  the  oxygen  removed. 

Two  types  of  reductase  have  been  recognized  and  are  dif- 
ferentiated by  their  action  on  methylene  blue.  One  type, 
which  appears  to  be  of  cellular  origin  and  is  present  in  fresh 
milk,  rapidly  decolourises  methylene  blue  solutions  in  the 
presence  of  a  trace  of  formaldehyde,  whilst  the  other  is  capable 
of  effecting  the  reduction  in  the  absence  of  formaldehyde  and 
is  of  bacterial  origin. 

BIOLOGICAL 

Immune  Bodies.  Although  the  examination  of  milk  for  the 
presence  of  immune  bodies  is  but  infrequently  required  in  con- 
nection with  public  health  work,  a  general  consideration  of 
these  bodies  and  their  significance  is  of  interest.  Before  con- 
sidering these  in  detail  it  will  be  advisable  to  review  briefly  the 
theory  of  immunity. 

After  an  attack  of  disease-producing  organisms,  animals 
usually  possess,  for  a  varying  length  of  time,  an  immunity 
against  a  further  attack,  and  this  immunity  is  ascribed  to  the 
presence  of  substances  known  as  immune  bodies.  The  re- 
searches of  Ehrlich  and  others  have  established  that  these 
immune  bodies,  or  anti-bodies  as  they  are  generally  described, 
are  produced  by  external  agencies.  In  addition  to  living  and 
dead  bacteria,  other  substances  such  as  animal  and  vegetable 
proteins,  animal  cells,  and  toxins,  may  act  as  antigens.  Ehr- 
lich 's  theory  of  immunity  hypothecates  the  existence,  in  the 
molecules  constituting  both  the  antigen  and  body  cell,  of 
binding  groups  or  haptophoric  receptors  which  fit  "  as  a  key 
fits  the  lock  "  and  which  anchor  the  antigen  to  the  body  cell. 
In  the  case  of  toxins,  other  receptors  are  also  assumed  to  be 
present,  viz.,  toxophores,  which  are  responsible  for  the  toxic 
effects  produced  after  the  antigen  has  been  anchored  to  the  cell. 


IMMUNE  BODIES  25 

• 

The  cell  molecules  may  be  destroyed  as  the  result  of  this  com- 
bination or  it  may  be  stimulated  by  defensive  action  to  the 
production  of  receptors;  continued  excitation  results  in  the 
production  of  more  receptors  than  are  necessary  for  the  func- 
tions of  the  cell  and  it  is  assumed  that  these  receptors  are  set 
free  in  the  fluids  surrounding  the  cells,  and  that  they  possess 
a  greater  affinity  for  the  antigen  than  the  same  receptors  of  the 
cell  molecule.  These  free  receptors  constitute  the  antibodies. 
Three  varieties  of  antibodies  are  known. 

(1)  Uniceptors,  such  as  antitoxins,  which  are  regarded 

as    comparatively    simple    and    which    combine 
directly  with  the  antigen. 

(2)  Uniceptors,  which  have  an  enzyme-producing  group 

in  addition  to  the    haptophoric    receptor  (agglu- 
tinins,  precipitins). 

(2)  Amboceptors,  which  require  the  presence  of  a  third 
substance  before  combination  with  the  antigen  can 
be  effected;  this  third  substance  is  known  as  com- 
plement. 

Antigens,  and  uniceptors  produced  by  them,  are  specific 
in  their  action,  and  this  applies  equally  to  the  amboceptor- 
complement-antigen  system  of  the  third  order  of  receptors. 
For  instance,  tetanus  antitoxin  acts  on  tetanus  toxins  and  on 
no  others,  and  typhoid  serum  agglutinates  only  B.  typhosus. 
This  statement,  however,  is  not  absolutely  true,  as  antigens 
produced  by  allied  groups  of  organisms  possess  receptors  which 
are  common  to  all,  but  as  the  specificity  becomes  more  definite 
with  increased  dilution  of  the  antibody,  the  affinity  between 
the  specific  receptors  must  be  considered  to  preponderate. 
The  amboceptors  of  the  third  order  of  antibodies  also  show 
relative  rather  than  absolute  specificity. 

The  antibodies  generally  are  distinguishable  from  comple- 
ments by  their  resistance  to  heat.  The  uniceptors  and  ambo- 
ceptors are  thermostabile,  i.e.,  are  not  destroyed  by  heating  to 


26  CONSTITUENTS  OF  MILK 

6°  C.  for  thirty  minutes,  whereas  complement  is  destroyed  by 
this  treatment;  complement  is,  therefore,  thermolabile. 

Antibodies,  like  enzymes,  are  of  unknown  chemical  constitu- 
tion and  are  usually  designated  by  the  nature  of  the  action  pro- 
duced; thus,  antitoxins  neutralise  toxins,  cytolysins  dissolve 
animal  cells,  haemolysins  dissolve  erythrocytes,  bacteriolysins 
dissolve  bacteria,  agglutinins  agglutinate  cells  and  bacteria,  and 
precipitins  produce  precipitates  from  solutions. 

Immunity,  by  which  is  understood  the  existence  of  a  cer- 
tain resistance  toward  deleterious  influences,  may  be  either 
acquired  or  natural.  The  apparent  immunity  of  individuals, 
races,  and  species  to  various  diseases  under  normal  conditions 
is  known  as  natural  immunity,  and  very  little  is  known  of  the 
etiological  factors  involved.  Acquired  immunity  may  be  acci- 
dental, as  in  the  case  of  the  irnmunity  acquired  by  an  attack  of  a 
disease,  or  artificially  acquired  by  the  introduction  into  the 
system  of  either  antigens  or  antibodies.  When  antibodies  are 
employed,  the  immunity  is  but  of  short  duration  compared 
with  the  several  years  of  immunity  obtained  by  the  use  of  anti- 
gens. The  former  process  is  known  as  passive  immunity  and 
the  latter  as  active  immunity. 

When  antibodies  are  present  in  the  blood,  certain  quantities 
are  excreted  by  the  milk  glands  and  may  be  found  in  the  milk. 
Ehrlich  has  demonstrated  that  offspring  may,  through  suckling, 
obtain  a  passive  immunity  from  either  an  actively  or  passively 
immunised  mother.  The  antibody  content  of  milk  is  usually 
very  much  weaker  than  that  of  the  blood  from  which  it  is 
derived.  Uniceptors  of  the  second  order  are  also  transferable 
to  the  milk  and  may  be  less  than,  equal  to,  or  even  greater,  than 
the  amounts  found  in  the  blood.  The  evidence  regarding  the 
transfer  of  the  third  order  of  antibodies  is  somewhat  conflicting. 
Amboceptors  and  complement  derived  from  the  blood  may 
appear  in  the  milk,  but  this  is  unusual  and  various  experi- 
menters have  stated  that  complement  is  not  present  in  normal 
ripe  milk  except  in  minute  traces.  In  colostrum  and  milk 
derived  from  udders  affected  with  mastitis,  however,  both 


OPSONINS  27 

l 

amboceptor  and  complement  may  be  present.  The  applica- 
tion of  the  complement  fixation  test  for  the  detection  of  colos- 
trum is  only  of  scientific  interest  and  mastitis  can  be  much 
more  readily  detected  by  an  examination  of  the  sediment  of  the 
milk. 

Opsonins,  bodies  which  prepare  bacterins  for  phagocytosis, 
the  process  by  which  a  cell  (phagocyte)  absorbs  bacterins  and 
other  particulate  matter,  have  also  been  demonstrated  in  milk. 

It  is  possible  that  anaphylactins,  which  induce  the  phenome- 
non known  as  anaphylaxis  or  hypersensitiveness,  may  occur  in 
milk  as  it  has  been  shown  by  Otto  that  the  progeny  of  hyper- 
sensitised  guinea  pigs  were  anaphylactic  to  homologous  antigens. 
The  transmission,  however,  may  have  been  either  intrauterine 
or  through  the  milk.  Mention  might  also  be  made  of  the  bene- 
ficial effect  upon  children  suckling  from  mothers  being  treated 
with  "  606,"  although  whether  the  results  are  due  to  the  pass- 
age of  antibodies  or  arsenic  is  still  in  dispute.  Considering  the 
indubitable  proof  of  the  passage  of  various  classes  of  anti- 
bodies from  the  blood  stream  to  milk,  it  is  reasonable  to  assume 
that  aggressins,  bodies  which  inhibit  the  protective  power  of  the 
cells,  and  toxins  are  also  transferable.  This  hypothesis  has 
been  experimentally  established,  but,  like. the  antitoxins,  the 
amounts  found  in  the  milk  are  considerably  smaller  than  in  the 
blood.  If  it  is  assumed  that  the  gastro-intestinal  tract  of  infants 
is  penetrated  by  proteids,  the  question  of  the  transference  of 
toxins  assumes  practical  importance.  Even  in  individuals 
showing  severe  symptoms,  by  far  the  greater  part  of  the  antigen 
is  anchored  to  the  cell  leaving  but  little  in  the  free  or  labile 
condition  in  the  system,  and,  as  only  a  fraction  of  this  is  trans- 
ferred to  the  milk,  the  total  amount  assimilated  by  the  off- 
spring is  probably  negligible;  a  posteriori  observations  confirm 
this  deduction. 

Since  milk  contains  various  proteid  substances,  it  is  capable 
of  acting  as  antigen  and  on  injection  produces  a  number  of 
antibodies.  The  lactoserum  obtained  by  the  use  of  cows' 
milk  contains  precipitins,  amboceptors,  and  hsemolysins,  which 


28  CONSTITUENTS  OF  MILK 

are  specific  in  their  reactions  and  may  be  used  as  qualitative 
tests  for  milk.  Bauer  succeeded  in  detecting  as  small  a  quan- 
tity as  1  c.cm.  of  cows'  milk  per  litre  of  human  milk  by  the 
complement  fixation  method.  The  various  proteids  of  milk, 
caseinogen  and  albumin,  etc.,  also  produce  specific  antibodies 
which  may  be  recognised  by  the  precipitin  method.  The 
specificity  of  lactoserum,  like  those  of  sera  in  general,  is  relative 
rather  than  absolute,  the  lactosera  of  closely  related  animals 
being  differentiated  by  the  intensity  of  the  reactions.  The 
phenomenon  of  anaphylaxis  may  also  be  induced  by  the  injec- 
tion of  milk.  Arthus  and  Besredka  state  that  boiled  milk,  as 
well  as  the  raw  product,  is  capable  of  producing  the  requisite 
conditions,  though  Miessner  found  that  a  larger  number  of 
injections  were  necessary  before  sensitisation  was  satisfac- 
torily established.  Caseinogen  and  albumin  also  produce 
specific  anaphylactins  which  may  be  used  as  a  basis  for  differ- 
ential tests. 

PhysicaL  The  characteristic  appearance  of  milk  is  pro- 
duced by  the  colloidal  suspension  of  caseinogen  complex  and 
the  emulsion  of  fat  globules.  When  milk  is  allowed  to  remain 
quiescent,  the  fat  globules,  being  of  smaller  density,  rise  to  the 
surface  and  form  a  layer  of  cream  which  is  distinctly  yellowish 
in  tint,  the  residual  milk  being  bluish  white  in  colour.  The 
opacity  is  diminished  by  the  addition  of  alkali,  which  dissolves 
the  caseinogen,  and  is  increased  by  any  process  that  reduces  the 
size  of  the  fat  globules.  Heat  alone,  at  different  temperatures, 
is  capable  of  reducing  the  diameter  of  the  fat  globules,  but  it 
may  be  more  effectively  accomplished  by  forcing  milk  heated 
to  60°  C.  through  very  small  orifices  under  high  pressure. 

The  specific  gravity  of  milk  bears  a  definite  relation  to  the 
total  solids  it  contains  (see  p.  70),  being  decreased  by  the  fat 
content  and  increased  by  the  solids  other  than  fat.  The  specific 
gravity  or  density  varies  considerably  with  variations  in  season, 
period  of  lactation,  breed,  and  character  and  quantity  of  food, 
but  1026.4  to  1037.0  (waterif^-§;=1000)  may  be  regarded  as 
the  extreme  limits.  When  milk,  freshly  drawn  from  the  udder, 


PHYSICAL 


29 


is  allowed  to  stand  for  one  hour  to  eliminate  air  bubbles,  it 
will  be  found  to  have  a  density  somewhat  lower  than  that 
taken  subsequently  (Recknagel's  phenomenon).  This  pecu- 
liarity has  been  investigated  by  several  observers.  Vieth  con- 
firmed Recknagel's  results  and  found  the  average  rise  to  be 
+1.3°  (water  =1000).  H.  Droop  Richmond33  reports  that  in 
70  per  cent  of  his  experiments  the  rise  varied  from  0.3°  to  1.5°, 
averaging  0.6°,  and  that  in  30  per  cent  of  the  observations  no 
rise  in  density  was  indicated;  also  that  the  rise  was  more 
rapid  at  low  temperatures  than  at  high  temperatures.  H.  D. 
Richmond,  from  consideration  of  experiments  made  in  con- 
junction with  S.  O.  Richmond  on  the  effect  of  heat  upon  the 
density  and  specific  heat  of  milk,  regards  the  phenomenon  as 
largely  due  to  the  increase  in  density  of  the  fat  on  solidification. 
Changes  in  the  milk  sugar,  cessation  of  expansion  of  the  case- 
inogen,  absorption  of  gases,  and  enzyme  action  have  also  been 
suggested  as  causes  of  this  phenomenon  but  cannot  be  regarded 

TABLE  VIII 
EFFECT  OF  TEMPERATURE  ON  VOLUME 


Temperature  in 
Degrees  Fahrenheit. 

Volume. 

Temperature  in 
Degrees  Fahrenheit. 

Volume. 

31 

1.00000 

60 

1.00229 

35 

1.00016 

65 

1.00298 

40 

1.00041 

70 

1.00372 

45 

1.00074 

75 

1.00451 

50 

1.00114 

80 

1.00549 

55 

1.00164 

as  satisfactory.  Various  data  confirming  Richmond's  hypoth- 
esis were  obtained  by  Toyonaga,  and  Fleishmann  and  Weig- 
ner.34  The  latter  observers  found  that  the  change  in  density 
was  proportional  to  the  amount  of  butter  fat  present.  Micro- 
scopical examinations  also  showed  that  the  solidified  globules 
were  of  greater  density  than  the  liquid  globules  at  the  same 
temperatures. 


30  CONSTITUENTS  OF  MILK 

Although  milk  contains  considerable  quantities  of  water 
(85-90  per  cent),  the  maximum  density  is  found  at  a  tempera- 
ture near  to  the  freezing  point  and  not  at  4°  C.  as  in  the  case  of 
water.  The  changes  in  the  volume  of  milk  due  to  temperature 
alterations  are  somewhat  variable,  being  dependent  upon  the 
composition;  the  preceding  table,  due  to  Richmond,  shows  the 
expansion  in  glass  of  milk  containing  3.8  per  cent  of  fat  and 
having  a  density  of  1032.0. 

The  viscosity  of  milk,  according  to  Taylor,35  is  not  propor- 
tional to  the  percentage  of  total  solids,  but  is  a  function  of  the 
fat  and  the  solids-not-fat  content.  He  found  that  the  relation 
is  expressed  by  the  formula: 

TJ       A  r  A     (viscosity— fat  percentage  X  0.0665) 
percentage  solids-not-fat  =  —          — ^       —  — -, 

and  that  the  viscosity  temperature  coefficient  was 

nt  =  ^+0.00723*  -  0.000156*2. 

Taylor's  determinations  of  the  viscosity  of  milk  raised  from  20° 
to  60°  C.  and  subsequently  cooled,  support  the  hypothesis  of 
Richmond  regarding  the  explanation  of  RecknageFs  phenome- 
non. Weigner36  found  that  homogenisation  of  milk  slightly 
increased  the  viscosity.  Two  samples  having  viscosities  of 
1.941  and  1.862,  as  determined  with  an  Oswald  viscosimeter, 
were  increased  by  homogenisation  to  1.967  and  1.889,  respec- 
tively. Weigner  thought  that  this  was  caused  by  increased 
adsorption,  especially  of  caseinogen. 

The  freezing  point  of  milk  is  slightly  lower  than  that  of  water, 
being  usually  —0.54  to  —0.57°  C.  and  is  especially  influenced 
by  the  mineral  content  other  than  that  associated  with  the 
caseinogen.  As  the  salts  are  not  subject  to  wide  variation  in 
the  milk  of  healthy  cattle,  the  freezing  point  is  usually  fairly 
constant.  This  forms  the  basis  of  the  cryoscopic  methods  for 
the  detection  of  milk  adulteration.  Aitkens37  shows  that  a 
consideration  of  the  osmotic  pressure  of  the  blood  of  animals 
and  that  of  the  milk  secreted  points  to  the  conclusion  that  the 


PHYSICAL 


31 


freezing  point  of  milk  will  never  fall  below  that  of  blood.  He 
found  the  freezing  point  of  the  blood  of  the  cow  to  be  —0.62°  C. 
and  that  of  cows'  milk  0.55°  C.±0.06°  C. 

In  contrast  with  the  relative  constancy  of  the  depression  of 
freezing  point  of  cows'  milk,  the  specific  conductivity  shows 
greater  variations,  although  milk  produced  under  normal  con- 
ditions does  not  show  very  marked  differences. 

The  following  results  are  given  by  various  observers: 

TABLE  IX 
CONDUCTIVITY  OF  MILK 

Koeppe  (1898) K  at  25°  C.=  0.00430-0. 00560 

Lehnert  (1897) 0.00487-0.00551 

Schnorf  (1905) 0.00485 

Benaghi  (1910) 0.00494-0.00517 

Jackson  and  Rothera  (1914) 0.00493-0.00641 

Jackson  and  Rothera  Herd  milk  (1914) .  . .  =0.00549-0.00587 

Jackson  and  Rothera  38  point  out  that,  owing  to  the  osmotic 
pressure  of  milk  being  controlled  by  that  of  the  blood,  the  sub- 
stances chiefly  responsible  for  this  manifestation,  viz.,  the 
milk  sugar  and  soluble  salts,  cannot  vary  independently,  but 
must  be  inter-related.  If  the  lactose  is  high  the  salts  must  be 
low,  and  conversely,  if  the  lactose  is  low  the  salts  must  be  high 
or  the  osmotic  pressure  would  be  lower  than  normal.  Jackson 
and  Rothera  found  experimentally  that  the  electrical  conduc- 
tivity of  milk,  which  is  mainly  due  to  the  soluble  salts,  is  in- 
versely proportional  to  lactose  content.  This  inverse  propor- 
tionality was  especially  observable  in  milk  produced  under 
pathogenic  conditions,  as  shown  by  the  following  example: 


Depres- 

Conduc- 

Lactose, 

sion  of 

Sol.  Ash, 

Insol.  Ash, 

Quarter. 

tivity,  K 

Per  Cent. 

Freezing- 

Per  Cent. 

Per  Cent. 

point.  A 

Left  anterior  (abnormal). 

0.0114 

1.50 

0.580 

0.615 

0.440 

Right  anterior  (normal)  . 

0.00569 

5.40 

0.575 

0.285 

0.625 

32  CONSTITUENTS  OF  MILK 

As  the  proteins  of  milk  obstruct  the  carriage  of  electricity 
by  the  moving  ions,  the  conductivity  of  whey  or  of  serum  is 
greater  than  that  of  the  milk  from  which  it  is  prepared.  Each 
1  per  cent  of  protein  reduces  the  conductivity  by  2.75  per  cent 
(Rothera  and  Jackson).  The  surface  tension  of  milk  is  lower 
than  that  of  water,  0.053  as  against  0.075  and  the  specific  heat 
of  milk  containing  3.17  per  cent  of  fat  is,  according  to  Fleish- 
mann,  0.9457. 

The  refractive  index  of  milk  cannot  be  determined  on  account 
of  its  opacity,  but  that  of  the  serum,  after  removal  of  the  case- 
inogen  and  fat,  has  been  determined  on  a  large  number  of  sam- 
ples by  various  observers  and  is  now  regarded  as  a  valuable 
aid  in  the  detection  of  adulteration  by  the  addition  of  water. 

This  method  is  of  special  value  on  account  of  the  removal 
of  the  constituents  of  milk  that  are  most  variable  in  amount, 
viz.,  fat  and  caseinogen,  leaving  a  serum  containing  the  lac- 
tose, mineral  matter,  and  albumin  which  are  generally  the  least 
variable.  Various  methods,  which  vary  somewhat  in  the 
completeness  of  precipitation  of  caseinogen  attained,  have  been 
employed,  39>40  and  normal  values  established  for  each.  The 
refractive  index  of  fresh  milk  serum,  prepared  by  filtration 
through  porous  plates,  varies  from  (ju£>20°  C.)  1.34200  to 
1.34275.  The  specific  gravity  of  milk  serum  is  equally  as  valua- 
ble as  the  refractive  index  (see  p.  79)  but  on  account  of  the 
longer  time  required  for  its  determination  it  is  not  generally 
used  as  a  routine  method.  The  ash  of  the  serum  also  affords 
valuable  information  for  the  detection  of  added  water.  (Lyth- 
goe,40  and  Burr  and  Berberich.41). 

BIBLIOGRAPHY 

1.  Van  Slyke  and  Bosworth.     Bull.  26,  N.  Y.  Expt.  Sta.     Geneva,  1912. 

2.  Schryver.     Proc.  Roy.  Soc.,  B.  86,  460-481. 

3.  Richmond.     Dairy  Chemistry.     London,  1914,  p.  30. 

4.  Soldner.     Landw.  Versuch.  Stat.     1888,  35,  351. 

5.  Lacquer  and  Sackur.     Beitr.  Chem.  Phys.  u.  Path.     1902,  3,  193. 

6.  Robertson.     Jour.  Phys.  Chem.     1911,  15,  179. 

7.  Osborne.     Zeit.  Physiol.  Chem.     1901,  33,  240. 


BIBLIOGRAPHY  33 

8.  Long.     Jour.  Amer.  Chem.  Soc.     1907,  29,  1334. 

9.  L.  L.  Van  Slyke  and  D.  D.  Van  Slyke.   Jour.  Amer.  Chem.  Soc.,  1907, 

38,  383. 

10.  Van  Slyke  and  Bosworth.    Bull.  37,  N.  Y.  Expt.  Sta.    Geneva,  19L4. 

11.  Cameron  and  Hurst.     Jour.  Amer.  Chem.  Soc.     1904,  26,  905. 

12.  Van  Slyke  and  Bosworth.     J.  Bio.  Chem.     1915,  20,  135. 

13.  Heintz.     Jour.  f.  Prakt,  Chem.  n.  F.     6,  33. 

14.  Hammerstein.    Maly's  Jahresb.    1872, 1118,  ibid.  1874, 135;  ibid.  1877, 

158. 

15.  Schmidt.     Beitrage  zur  Kenntniss  der  Milch.     Dorpat,  1871. 

16.  Loevenhart.     Zeit.  f.  Physiol.  Chem.     1904,  41,  177. 

17.  Briot.     Etudes  sur  la  pressure  et  1'antipressure.     Th6se  de  Paris,  1900. 

18.  Harden  and  Macallum.     Biochem.  Jour.     1914,  8,  90. 

19.  Duclaux.     Traite"  de  Microbiologie.     Paris,  1899,  II,  291. 

20.  Van  Slyke  and  Bosworth.     Jour.  Biol.  Chem.     1913,  14,  203. 

21.  Geake.     Biochem.  Jour.     1914,  8,  30. 

22.  Van  Slyke  and  Hart.     J.  Amer.  Chem.  Soc.     1905,  33,  461. 

23.  Liwschiz.     Diss.  Miinchen.     1913.     Z.  Kinderheilk,  Ref.  8,  345. 

24.  Hewarden.     Zeit.  f.  Physiol,  Chem.     1907,  52,  184. 

25.  Morgenrath.     Centr.  f.  Bakt.  Abt.  I,  26,  271. 

26.  Lindet.     Bull.  Soc.  Chim.     13,  929. 

27.  Bechamp.     Compt.  Rendus.     96,  1508. 

28.  Babcock  and  Russell.     Centr.  f .  Bakt.  u.  Par.,  Abt.  II,  1900, 6, 17-22. 

and  79-88. 

29.  Wender.     Oesterr.  Chem.  Zeit.     6,  13. 

30.  Marfan  and  Gillet.     Monatschr.  f.  Kinderheilk.     1902,  I,  57. 

31.  Nobe"court  and  Merklen.     Compt.  Rend.  Soc.  Biol.     1901,  53,  148. 

32.  Kastle  and  Porch.     Jour.  Bio.  Chem.     1908,  4,  301. 

33.  Richmond.     Dairy  Chemistry.     London,  1914,  p.  76. 

34.  Fleishmann  and  Weigner.     Jour.  Landw.     61,  283. 

35.  Taylor.     J.  Proc.  Roy.  Soc.  N.  S.  W.     47,  II,  174. 

36.  Weigner.     Kolloid.  Z.     1914,  15,  105. 

37.  Aitkens.     Chem.  News.     1908,  97,  241. 

38.  Jackson  and  Rothera.     Biochem.  Jour.     1914,  8,  1. 

39.  Arb.  Gesundheits.     40.     Heft.     3. 

40.  Lythgoe.     J.  Ind.  and  Eng.  Chem.     6,     904. 

41.  Burr  and  Berberich.    Chem.  Zeit.,    32,  617. 


CHAPTER  II 


THE  NORMAL  COMPOSITION  OF  MILK 

THE  average  composition  of  cows'  milk  as  compared  with 
the  milk  of  various  other  mammals  is  shown  in  Table  No.  X. 
(Bunge !). 

TABLE  X 

COMPOSITION  OF  MAMMALS'  MILK 


Fat. 

Caseinogen. 

Albumin. 

Lactose. 

Ash. 

Human  (1)  

3  1 

5  9 

0  2 

Human  (2) 

3  8 

1  2 

0  5 

6  0 

0  2 

Human  (3)  

3  3 

6  5 

0  3 

Dog 

12  5 

5  2 

1  9 

3  5 

1  3 

Cat 

3  3 

3  1 

6  4 

4  9 

0  6 

Rabbit  

10  5 

2  0 

2  6 

Guinea  Dig 

45  8 

1  3 

0  6 

Sow  

6  9 

3  8 

1.1 

Elephant  
Horse. 

19.6 
1  2 

1  2 

0  8 

8.8 
5  7 

0.7 
0  4 

Ass  

1  6 

0  7 

1.6 

6.0 

0.5 

Cow   

3  7 

3  0 

0  9 

4  9 

0  7 

Goat. 

4  8 

3  2 

1  i 

4  5 

0  8 

Sheep  

6  9 

5  0 

1.6 

4.5 

0.9 

Reindeer  

17  1 

8  4 

2.0 

2  8 

1  5 

Camel 

3  1 

5  6 

1  8 

Llama  

3  2 

3  0 

0.9 

5.6 

0.8 

Porpoise 

54  8 

7  6 

0  5 

Apart  from  the  very  varying  amounts  of  fat  the  similarity 
in  the  composition  of  the  milk  of  these  various  mammals  is  very 
remarkable. 

34 


AVERAGE  COMPOSITION 


35 


Various  observers  have  recorded  the  results  of  thousands 
of  analyses  of  cows'  milk  and  some  of  the  most  authentic  are 
given  in  Table  XI. 

TABLE  XI 
COMPOSITION  OF  COWS'  MILK 


Average  of 

Water. 

Fat. 

Casei- 
nogen. 

Albu- 
min. 

Lac- 
tose. 

Ash. 

280,000  analyses,   Aylesbury  Dairy 

Co.,  London,  Richmond  

87.35 

3.74 

3.0 

0.4 

4.70 

0.75 

5552  analyses  in  U.  S.  A.     Van  Slyke 

87.10 

3.90 

2.5 

0.7 

5.10 

0.70 

Cheese    factory    milk.     New    York 

State.     May  to  Nov.     Van  Slyke  . 

87.40 

3.75 

2.45 

0.7 

5.00 

0.70 

800  analyses  by  Koenig  

87.27 

3.64 

3.02 

0.53 

4.88 

0.71 

The  essential  difference  between  the  European  and  Amer- 
ican results  lies  in  the  ratio  of  lactose  to  proteids  and  the  rela- 
tive amounts  of  caseinogen  and  albumin  that  make  up  the  total 
proteids.  Numerous  analyses  by  the  author  of  Canadian  milk 
show  that  the  average  ratio  of  lactose  to  proteid  in  that  country 
is  distinctly  higher  than  those  recorded  by  Richmond  and 
Koenig.  The  figures  of  Lythgoe2  for  milk  in  Massachusetts, 
confirm  this  view.  At  least  a  portion  of  the  differences  between 
the  relative  amounts  of  caseinogen  and  albumin  in  the  analyses 

TABLE  XII 
MAXIMUM  VARIATIONS  IN  COMPOSITION 


Fat. 

Solids  Not-fat. 

Maximum         

Per  cent. 

14.67 

Per  cent. 

13.76 

Minimum                    

1.04 

4  90 

recorded  in  the  above  table  is  probably  due  to  errors  in  the 
various  methods  used  for  the  determination  of  these  constit- 


36 


THE  NORMAL  COMPOSITION  OF  MILK 


pq 

fo 

c 

h 
b 


c 

1 

OS  '1-1  00  O  CO  <N 

CO          O                                         iO 

00          O             OJ   cO  CO          OS 

-  •§ 

i-t  CO  00  Tj<  <N  O 

T-H 

O               i-H                   |>     i-H     O 

ll 

Qffl 

iO  CO  O  CO  CO  O 
i—l  IO  IO  OS  OS  t>» 

<M  CO  00  •"*!  C^  O 
1—  1 

CO           b- 

00           CO              CO   00 

O           i—  i              00   CQ 

!j 

<M  IO  b-  *O  iO  CO 

CO  OS  CO  CO  C^  t>- 

(N  CO  00  rf  CO  O 
1-H 

co  "tf  Tt<        o 

IS 

£ 

-*  i—  I  CO  OO  OS  CO 
CO  O  CO  00  OS  t>- 

(M-  rj^  00  ^  <N  O 

10        co                               co 

b-           CO              b»   00   IO           »O 

O          I-H             l>i   (M    O 

CO    ^^    ^^            ^^ 

•  Jj 

I  I 

00  C^  CO  to  <N  iO 
OS  <N  b-  OO  W  b- 

<N  rt<  00  "^  CO  O 

CO           1-1                                         OS 
b-          10            OS  iO  OS          fr<- 

O          I-H             b-   (M   i-t          O 

«d 

S| 

O  O  r-  1  CO  O5  CO 

i—  t  <N  00  00  CO  b- 
CO  T^  00  ^  CO  O 

OS            CO                                              I-H 
b-           <<ti              O   b-    ^H           00 

o        i-i          06  oi  csi        o 

CO    '^    "^ 

Jb 

S  %  8  S  £  £ 

CO  -*'  GO  -*  CO  O 

CO 
*O           '•^                                           *O 
t^*           *O              ^^   t*"*   t^*           l>» 

O           1-"              00    CO   I-H           O 

|| 

"^  iO  O^  1s*  iO  iO 

Is-  CO  O  00  Tf  l>- 

co  ^  OS  ^  co  o 

t^           ^^              CO   OS   O^           00 

1* 

O  CO  b-  rf  CO  IO 
CO  (M  CO  00  l>  b- 

T}i  10  OS  rt  CO  O 

i—  i           O                                •              • 
b-           CO              C<l    OS       • 

o"        I-H          oo  co     • 

CO   '^       •       •       • 

! 

iO  iO  O  TfH  CO  (M 

b-  CO  »—  1  O5  Tf  t> 

Tt^  IO  OS  ^^  CO  ^^ 

1-H 

CO 
1-1        co                               oo 

CO           ^f              I-H   C^   l^-           !>• 

o             &    : 
o             2    : 
N             %   : 

1         3  ! 

S               SS 

V                                     .t2 

:      -§ 

o-Sc    gi: 

2  o  ^ 

•a  i|3 

-g  -^  a  «  o  ^3 

o'lc^^slft 

^  t2     £f26-<c»^ 

LIMITS  AND  VARIATIONS 


37 


uents.  Later  American  analyses  have  shown  that  the  normal 
albumin  content  of  0.7  per  cent,  as  recorded  by  Van  Slyke, 
is  too  high  and  that  0.5  per  cent  is  much  nearer  the  correct 
value. 

Limits  and  Variations.  The  variation  in  the  composition 
of  milk  obtained  from  herds  is  not  usually  very  great,  but  that 
of  individual  cows  may  vary  between  very  wide  limits.  The 
following  figures  show  the  maximum  and  minimum  that  have 
been  recorded,  the  former  by  Cook  and  Hills  of  milk  from  a 
Jersey  cow  just  before  going  dry,  and  the  latter  by  Richmond. 

The  fat  content  of  milk  is  very  variable  and  depends  upon  a 
number  of  factors,  the  chief  of  which  are  breed,  food,  season, 
interval  between  milkings,  and  stage  of  lactation. 

The  breed  of  the  cow  has  a  very  important  bearing  upon  the 
quality  of  the  milk  produced,  some  (Jersey  and  Guernsey)  giv- 
ing milk  containing  60  per  cent  more  fat  than  others  (Holstein). 
Results  of  analyses  of  milk  from  various  breeds  are  recorded 
in  Tables  XIII,  XIV,  and  XV. 


TABLE  XIV 
FAT  AND  SOLIDS  NOT-FAT  IN  MILK  FROM  VARIOUS  BREEDS 

(VlETH) 


Breed. 

\ 
TOTAL  SOLIDS. 

FAT. 

SOLIDS  NOT-FAT. 

Aver- 
age. 

Maxi- 
mum. 

Mini- 
mum. 

Aver- 
age. 

Maxi- 
mum. 

Mini- 
mum. 

Aver- 
age. 

Maxi- 
mum. 

Mini- 
mum. 

Dairy  shorthorn  .  . 
Pedigree     " 
Jersey 

12.90 
12.86 
14.89 
13.70 
13.22 
14.18 
12.61 
14.15 

18.70 

16.8 
19.9 
18.6 
16.2 
17.4 
16.1 
17.6 

10.2 
10.5 
11.0 
10.6 
9.7 
11.5 
10.2 
11.9 

4.03 
4.03 
5.66 
4.72 
4.34 
4.87 
3.59 
4.91 

10.2 

7.5 
9.8 
10.5 
6.6 
7.6 
6.5 
8.3 

1.3 
1.9 
2.0 
1.8 
2.5 
2.9 
1.4 
3.0 

8.87 
8.83 
9.23 
8.98 
8.88 
9.31 
9.02 
9.24 

10.6 
9.8 
10.4 
10.6 
10.2 
10.3 
10.0 
9.6 

7.6 
7.6 
8.1 
4.9 
7.1 
8.4 
7.9 
8.9 

Kerry  

Red  Polled  

Sussex 

Montgomery  
Welsh 

38 


THE  NORMAL  COMPOSITION  OF  MILK 


The  figures  in  Table  XV  are  compiled  from  results  published 
by  the  various  American  Experimental  Agricultural  Stations. 

TABLE  XV 


Breed. 

Total 
Solids. 

Fat. 

Lactose. 

Proteid. 

RATIO. 

Lactose 
Proteid  ' 

Proteid 
Fat    ' 

Jersey  

14.70 
14.49 
12.72 
12.00 
.   12.57 

5.14 

4.98 
3.85 
3.45 
3.63 
4.03 

5.04 
4.98 
5.02 
4.65 
4.89 

3.80 
3.84 
3.34 
3.15 
3.32 

1.32 
1.30 
1.50 
1.47 
1.47 

0.74 
0.77 
0.87 
0.91 
0.91 

Guernsey.  .  .  . 

Ayrshire  
Holstein  
Shorthorn  .... 
Red  Poll 

The  influence  of  breed  upon  the  chemical  characteristics  of 
the  fat  was  investigated  by  Eckles  and  Shaw  3  and  their  results 
are  summarised  in  Table  XVI. 


TABLE  XVI 
EFFECT  OF  BREED  ON  CHARACTERISTICS  OF  FAT. 

AND  SHAW) 


(ECKLES 


Breed. 

Relative  Size 
of  Fat 
Globules. 

Iodine 
Number. 

Saponifica- 
tion  Value. 

Reichert- 
Meisal 
Value. 

Melting- 
point, 
Centigrade. 

Jersey  

328 

30.5 

228.9 

26.7 

32.9 

Ayrshire. 

150 

31  6 

228  2 

25  9 

33  5 

Holstein  

142 

34.2 

229.1 

25.5 

32.9 

Shorthorn  

282 

34.4 

227.6 

26.3 

33.2 

It  is  evident  from  the  results  recorded  that  the  breed  of  cow 
has  a  marked  effect  upon  the  composition  of  the  milk  obtained 
and  that  certain  constituents  are  more  affected  than  others. 
The  fat  is  the  most  variable  constituent,  though  the  total 


LIMITS  AND  VARIATIONS  39 

amount  of  fat  yielded  by  the  various  breeds  is  far  less  so  and  is 
due  to  the  quantity  of  milk  being  usually  inversely  propor- 
tional to  the  fat  percentage  in  the  milk.  The  proportion,  how- 
ever, is  not  a  direct  one  and  it  has  been  proved  on  many  occa- 
sions that  the  breeds  giving  the  low  fat  percentages  yield  the 
largest  total  weight  of  fat.  For  this  reason  the  Dutch,  Frisian, 
and  Holstein  breeds  are  very  popular  for  dairy  purposes. 

Concerning  the  effect  of  food  upon  the  composition  of  milk, 
numerous  investigations  have  been  made  but  the  results  ob- 
tained are  apparently  somewhat  contradictory.  This  is  prob- 
ably partially  due  to  the  conditions  under  which  the  experi- 
ments were  conducted  being  not  strictly  comparable.  Earlier 
observers  failed  to  appreciate  the  fact  that  a  certain  weight  of 
fat,  proteid,  and  carbohydrates  is  necessary  for  providing  body 
heat  and  for  the  repair  of  waste  tissue  in  the  cow,  and  that  this 
amount  is  proportional,  though  not  directly  so,  to  the  weight  of 
the  animal.  If  the  food  ration  is  only  slightly  in  excess  of  this 
quantity,  the  effect  of  stimulants,  such  as  oil  cake,  would  be  to 
immediately  increase  both  the  percentage  and  total  quantity 
of  butter  fat  secreted;  on  the  other  hand,  if  the  ration  is  suf- 
ficient for  the  body  maintenance  and  milk  secretion,  additional 
food  would  probably  not  increase  either  the  percentage  or  the 
quantity  of  butter  fat,  and  it  is  conceivable  that  they  may  even 
be  somewhat  reduced  by  this  over-feeding  process. 

Of  the  more  reliable  investigations,  those  of  Morgen,  Beger, 
Fingerling,  Doll,  Hancke,  Sieglin,  and  Zielstorff4  might  be 
mentioned.  They  found  that  food  free  from  fat  sufficed  for  the 
maintenance  of  animals  in  a  healthy  condition  and  increased 
the  live  weight  of  the  animal,  but  was  totally  unsuitable  for 
milk  production.  The  addition  of  food  fat  in  quantities 
equivalent  to  0.5  to  1.0  gram  per  kilo  of  the  animal  weight 
favoured  the  production  of  milk  fat.  Later,  the  first  three 
observers,  in  a  series  of  experiments  extending  over  six  years, 
obtained  results  which  showed  that  of  all  foods,  fat  alone  exerts 
a  specific  action  on  the  production  of  milk  fat  and  that,  within 
certain  limits,  fat  is  the  most  suitable  food  for  butter  fat  pro- 


40 


THE  NORMAL  COMPOSITION  OF  MILK 


duction.  Malmejac  5  reports  the  following  comparative  figures 
obtained  in  Algeria  from  cattle  feeding  on  poor  and  rich 
forage. 


Poor  Dry  Grass. 

Rich  Forage. 

Total  solids  

11  62-14  25 

13  76-14  90 

Fat. 

3  33-  3  50 

4  05-  4  90 

Lactose  

4  53-  5  64 

4  47-  5  55 

Proteid  

3  13-  4  46 

3  33-  4  54 

Ash 

0  60-  0  90 

0  82-  0  93 

Brewery  and  distillery  waste  grains  in  the  wet  condition 
have  often  been  fed  to  cows  on  account  of  the  low  price  of  this 
material,  but  this  procedure  ultimately  proves  to  be  false 
economy,  as  both  the  relative  and  absolute  amount  of  milk  fat 
produced  is  reduced.  During  the  last  decade  there  has  been 
a  decided  tendency  towards  scientific  feeding  of  dairy  animals 
with  a  well  balanced  ration  which  is  just  sufficient  for  the  main- 
tenance of  body  weight  and  also  for  the  production  of  a  definite 
quantity  of  milk  containing  a  specified  amount  of  butter  fat. 
In  this  ration,  digestibility,  palatability,  and  proportion  of 
roughage  to  concentrates,  are  considered  and  calculated.  An 
example  of  this  rational  feeding  is  seen  in  the  herds  of  the 
Minnesota  Experimental  Station,  as  compared  with  the  other 
herds  of  the  state.  The  common  cows,  i.  e.,  cows  with  no  dairy 
heredity,  of  the  Experimental  Station  yielded  5000  Ibs.  of  milk 
equal  to  222  Ibs.  of  butter  per  head  as  against  4000  Ibs.  of  milk 
equal  to  175  Ibs.  'of  butter  per  head  for  the  whole  State  of 
Minnesota. 

Stable  or  byre  conditions,  fatigue,  and  temperature,  also 
have  slight  effects  upon  the  fat  content  of  the  milk  produced. 

The  seasonal  variation  in  the  amount  of  butter  fat  in  milk, 
according  to  Droop  Richmond's  figures,  is  well  marked  and 
always  occurs;  he  finds  that  the  fat  content  usually  decreases 
during  the  spring  and  summer  months,  reaching  a  minimum 
about  midsummer,  and  then  gradually  rises  to  a  maximum 


LIMITS  AND  VARIATIONS 


41 


during  the  winter.  The  fat  content  of  the  milk  of  Massa- 
chusetts and  Ontario  shows  several  modes  during  the  year, 
although  the  average  values  for  the  summer  months  are  less 
than  those  for  the  winter  months.  Diagram  No.  I  shows  the 
monthly  variations  in  England  for  1897-1913,  as  compiled 
by  Richmond,  in  Massachusetts  as  reported  by  Lythgoe  of 
State  Board  of  Health,  and  in  Ontario  as  calculated  from  the 
Ottawa  analyses  of  the  author.  Lythgoe  has  suggested  that 

DIAGRAM  No.  I 

EFFECT  OF  SEASON    ON   FAT  CONTENT 


4.1 


4.0 


3.8 


3.7 


3.C 


3.5 


p 

n—- 

—o*, 

/ 

*'    \ 

\ 

A 

p— 

A 

* 
^^ 

^ 

~*o 

s 

o 

o-' 

A 

V 

A 

\ 

/ 

~JL 

/- 

—  c^ 

^0 

•*-o 

\ 

X 

v' 

NX. 

V 

/ 

V 

/ 

X 

V 

J 

/ 

\ 

V 

/ 

England  . 
Massachusetts  
Canada  

Jan.    Feb.    Mar.    Apr.     May    June   July    Aug.    Sept.    Oct.     Nov.    Dec. 


the  irregularities  in  the  curve  for  the  Massachusetts  supply,  as 
compared  with  Richmond's  results,  are  due  to  the  larger  number 
of  samples  examined  by  the  latter.  Lythgoe's  results,  however, 
are  calculated  from  approximately  13,000  samples  examined 
during  three  years,  and  the  similarity  of  the  curve  to  that 
plotted  from  the  author's  analyses  of  over  9000  samples  sug- 
gests that  the  number  of  modes  in  the  curves  is  not  fortuitous, 
but  is  due  to  seasonal  variations  together  with  variations  caused 
by  changes  of  food  peculiar  to  local  climatic  conditions. 

Richmond  also  found  that  there  were  slight  daily  variations 


42  THE  NORMAL  COMPOSITION  OF  MILK 

in  the  quality  of  milk,  the  fat  content  of  Monday's  milk  being 
usually  slightly  lower  than  that  of  the  other  days,  but  this  is 
apparently  due  to  the  usual  intervals  between  milkings  being 
slightly  disturbed  during  the  week-end. 

The  intervals  elapsing  between  milkings  have  been  shown  by 
various  observers  to  have  an  influence  on  the  percentage  of 
fat,  though  relatively  little  on  the  absolute  amount.  Fleisch- 
mann6  found  morning  milk  slightly  richer  than  evening  milk 
and  decided  that  the  fat  content  varied  with  the  intervals 
between  milking.  Richmond  7  as  the  result  of  over  100,000 
analyses  made  during  sixteen  years,  gives  the  figures  for  the 
fat  content  of  morning  and  evening  milk  as  3.56  and  3.93  per 
cent,  respectively;  the  intervals  being  10.8  and  13.2  hours. 
His  results  also  show  that  the  difference  is  more  marked  during 
the  summer  months.  Eckles  and  Shaw  8  found  that  with  equal 
intervals  between  milkings,  the  morning  milk  was  slightly  higher 
in  fat  content  than  the  evening  milk.  The  Reichert-Meissl 
and  Koettstoffer  numbers  of  the  butter  fat  were  usually  lower, 
and  the  iodine  number  usually  higher  in  the  evening  milk,  while 
no  appreciable  constant  variation  could  be  detected  in  the 
physical  characteristics.  With  animals  milked  more  than 
twice  daily,  the  variations  in  the  fat  content  of  the  milk  were 
larger  and  the  highest  value  was  usually  found  in  the  milk 
drawn  near  the  middle  of  the  day.  The  explanation  of  this  is 
probably  connected  with  the  interval  between  feeding  and 
milking. 

The  influence  of  the  stage  of  lactation  upon  the  fat  content 
of  milk  has  been  the  subject  of  much  experimental  work,  and 
although  some  of  the  data  is  slightly  contradictory,  it  has  been 
generally  established  that  the  percentage  of  fat  usually  de- 
creases during  the  first  three  months  of  lactation,  then  remains 
fairly  constant  for  four  to  five  months,  and,  finally,  rises  rapidly 
to  a  maximum.  This  process  is  well  illustrated  by  the  results 
of  Eckles  and  Shaw  9  which  are  given  in  Table  XVII. 

The  chemical  and  physical  characteristics  of  the  butter  fat 
obtained  in  these  experiments  are  recorded  in  Table  XVIII. 


LIMITS  AND  VARIATIONS 


43 


TABLE  XVII 
AVERAGE  PERCENTAGE  OF  FAT  BY  FOUR-WEEK  PERIODS 


Jeisey. 

Shorthorn. 

Ayrshire. 

Holstein. 

First  period  

5  20 

4  08 

3.94 

3.14 

Second 

4  91 

3  88 

3.68 

2.87 

Third           

5.02 

3.71 

3.60 

2.78 

Fourth         

4  79 

3  54 

3.59 

3.11 

Fifth 

4  88 

3  56 

3  70 

3.11 

Sixth            

4  98 

3  58 

3.52 

2.98 

Seventh 

4  93 

3  69 

3  63 

3.03 

Eighth 

4  83 

3  73 

3  74 

3  09 

Ninth           

4  84 

4  19 

3.71 

3.05 

Tenth 

4  88 

4  19 

4  05 

3  31 

Eleventh      

5.23 

4.11 

4.92 

3.39 

Twelfth        

5  68 

3.96 

3  70 

Thirteenth 

5  48 

4  18 

4  48 

Fourteenth  

6.47 

3.68 

TABLE  XVIII 
RELATION  OF  LACTATION  PERIOD  TO  CHEMICAL  AND 

PHYSICAL  CONSTANTS  OF  FAT 
AVERAGE  DETERMINATIONS  BY  FOUR-WEFX  PERIODS 


Period. 

Percentage 
of  Fat. 

Relative 
Size  of 
Globules. 

Melting 
point, 
Centigrade 

Iodine 
Number. 

Reichert- 

Meissl 
Number. 

Sapomfica- 
tion 
Number. 

1 

4.00 

357 

31.7 

33.3 

29.1 

223.7 

2 

3.85 

307 

32.9 

31.6 

27.5 

230.4 

3 

3.79 

249 

32.8 

32.2 

27.1 

231.0 

4 

3.77 

256 

33.1 

30.8 

26.4 

229.6 

5 

3.82 

200 

33.3 

31.4 

26.6 

229.2 

6 

3.79 

204 

33.2 

31.7 

26.4 

228.9 

7 

3.83 

201 

33.3 

32.9 

25.5 

225.7 

8 

3.85 

192 

33.4 

33.3 

22.2 

226.7 

9 

3.97 

180 

33.5 

34.6 

24.2 

225.6 

10 

4.11 

152 

33.9 

35.4 

22.5 

223.4 

11 

4.22 

162 

34.7 

35.5 

22.2 

223.8 

12 

4.54 

166 

33.8 

35.2 

20.3 

220.6 

13 

4.66 

110 

36.5 

39.2 

17.2 

216.6 

44 


THE  NORMAL  COMPOSITION  OF  MILK 


It  was  found  that  the  size  of  the  globules  at  the  commence- 
ment of  lactation  was  about  twice  the  average  size  for  the  whole 
period;  the  size  sharply  diminished  during  the  first  six  weeks 
and  then,  after  remaining  fairly  constant  for  some  months, 
rapidly  declined.  The  iodine  value  varied  directly  with  the 
fat  content,  and  the  saponification  value,  after  a  preliminary 
rise,  declined  slowly,  but  gradually,  with  the  constantly  decreas- 
ing proportion  of  volatile  fatty  acids.  The  melting  point 
remained  comparatively  steady  until  the  last  periods  when  a 
perceptible  rise  occurred;  the  refractive  index  showed  no  appre- 
ciable variations. 

Non-fatty  Solids.  The  non-fatty  solids  of  milk  are  subject 
to  variations  from  causes  similar  to  those  which  determine 
the  variation  in  the  fat  content.  The  influence  of  breed  is 
shown  in  Tables  XIII,  XIV,  and  XV,  and  that  of  season  in 
Table  XIX  (Richmond  10). 

TABLE  XIX 
INFLUENCE  OF  SEASON  ON  SOLIDS-NOT-FAT 


Month. 

Fat. 

Solids- 
not-fat. 

Lactose. 

Proteid. 

Ash. 

January 

3  80 

8  95 

4.62 

3  57 

0  76 

February  

3.70 

8.97 

4.70 

3.52 

0.75 

March 

3  62 

9.91 

4.72 

3  45 

0  74 

April 

3  62 

8  83 

4  66 

3  42 

0  75 

May  

3.47 

8.85 

4.64 

3.47 

0.74 

June 

3  44 

8  82 

4  68 

4  42 

0  74 

July  .  . 

3.59 

8.67 

4.69 

3.23 

0.75 

August  

3.72 

8.55 

4.59 

3.25 

0.71 

September. 

3  88 

8  63 

4  63 

3  25 

0  74 

October 

3  91 

8  76 

4  63 

3  38 

0  75 

November  

3.94 

8.81 

4.63 

3.42 

0.76 

December                 .    .  . 

3.80 

8.81 

4.56 

3.50 

0.75 

These  results  show  that   the  solids-not-fat  decline  sympa- 
thetically with  the  fat  during  the  spring  and  summer  months, 


NON-FATTY  SOLIDS  45 

and  increase  during  the  autumn  and  winter  seasons.  The  sep- 
aration of  the  constituents  forming  the  non-fatty  portion  of 
solids  makes  it  apparent  that  the  decrease  in  the  summer 
months  is  due  chiefly  to  the  smaller  proteid  content,  the  lac- 
tose and  ash  remaining  fairly  constant.  The  author's  results 
for  Ottawa  milks  also  show  a  tendency  towards  a  decline  in  the 
non-fatty  solids  during  the  summer  months,  though  the  varia- 
tions are  more  irregular  than  in  the  series  of  Richmond  given 
above.  In  these  results  the  proteid  was  also  the  greatest  variant 
and  usually  accompanied  the  variations  in  the  fat  content. 

Richmond n  found  no  difference  between  the  non-fatty 
solids  of  evening  and  morning  milk  on  calculating  the  average 
results  for  a  number  of  years.  Eckles  and  Shaw  8  also  found 
no  appreciable  difference  in  the  total  amount  of  non-fatty  solids 
in  morning  and  evening  milk,  but  their  results  show  that  this 
is  due  to  an  increase  of  proteid  in  the  morning  milk  with  an 
equivalent  reduction  in  the  lactose  content. 

The  effect  of  the  stage  of  lactation  upon  the  solids-not-fat 
has  also  been  reported  upon  by  Eckles  and  Shaw.9  The  lac- 
tose remained  comparatively  constant  (vide  Table  XIX)  during 
the  greater  portion  of  the  period  with  a  slight  decline  during 
the  last  two  to  three  months.  The  ash  was  constant  and  the 
proteid  decreased  and  increased  sympathetically  with  the  fat 
though  not  in  direct  proportion  to  it. 

This  sympathetic  relation  between  the  amount  of  fat  and 
proteid  in  milk  has  led  to  the  introduction  of  several  formulae 
for  the  calculation  of  the  proteid  content  from  that  of  the  fat. 
Timpe  suggested  the  formula  P  =  2+0.35F  in  which  P  and  F 
represent  the  percentages  of  proteid  and  fat,  and  gave  many 
analyses  in  support  of  it,  but  Richmond  has  pointed  out  that 
when  the  series  is  extended,  the  agreement  practically  disap- 
pears. Van  Slyke's  formula12  P  =  0.4(^-3.0)  +2.8,  is  to  be 
preferred  to  that  of  Timpe  but  cannot  be  considered  as  entirely 
satisfactory.  These  formulae  are  calculated  from  the  averages 
of  many  analyses^  and  represent  the  average  relation  between 
fat  and  proteid  in  normal  milk.  Whilst  this  is  of  considerable 


46 


THE  NORMAL  COMPOSITION  OF  MILK 


TABLE  XX 

INFLUENCE   OF  STAGE   OF   LACTATION   ON   COMPOSITION 
BY  FOUR-WEEK  PERIODS 


Period  No. 

Fat. 

Lactose. 

Proteid. 

Total  Solids. 

1 

4.00 

4.87 

2.68 

12.74 

2 

3.85 

4.84 

2.36 

12.26 

3 

3.79 

4.94 

2.49 

12.29 

4 

3.77 

4.82 

2.49 

12.24 

5 

3.82 

4.80 

2.62 

12.35 

6 

3.79 

4.75 

2.68 

12.50 

7 

3.83 

4.88 

2.68 

12.61 

8 

3.85 

4.83 

2.74 

12.70 

9 

3.97 

4.62 

2.87 

12.78 

10 

4.11 

4.55 

3.06 

13.16 

11 

4.22 

4.74 

3.19 

13.46 

12 

4.54 

4.91 

3.38 

14.04 

13 

4.66 

4.70 

3.64 

14.23 

14 

5.08 

5.01 

3.70 

15.29 

scientific  interest,  it  is  of  comparatively  little  value  to  the  milk 
examiner  who  is  required  to  give  an  expression  of  opinion  upon 
analytical  results  with  reference  to  sophistication.  Such  sam- 
ples may  be  derived  from  many  sources  and  their  composition 
influenced  by  many  factors  concerning  which  he  has  little  or 
no  information;  the  examiner  is,  therefore,  more  vitally  inter- 
ested in  the  natural  variations  from  the  average  than  in  the 
average  itself. 

Reference  has  previously  been  made  to  various  factors  which 
produce  variation  in  the  composition  of  milk  but  it  is  advisable 
to  discuss  in  more  detail  their  effect  upon  the  relative  propor- 
tions of  the  various  constituents.  The  effect  of  breed  upon  the 

proteid        ,    lactose      , .  -,1,1  f  f  , 

t—j and  TT  ratios  together  with  the  percentage  of  fat 

fat  proteid 

in  the  total  solids  is  shown  in  tables  XXI,  XXII,  and  XXIII. 

Although  some  of  these  results  are  somewhat  discordant, 
the  general  tendency  is  usually  in  the  same  direction.  When 


NON-FATTY  SOLIDS 


47 


TABLE  XXI 
PROTEID 


FAT 


RATIO 


Breed. 

Van  Slyke. 

Lythgoe. 

Eckles  and 
Shaw. 

New  Jersey 
Expt.  Stat. 

Holstein.     Frisian  
Dutch  belt  .             .    . 

0.87 

0.86 
0  83 

0.95 

0.93 

Ayrshire  

0.82 

0.75 

0.88 

0.93 

American  Holderness  
Shorthorn 

0.83 
0  80 

0  96 

0  89 

Devon  

0.80 

Guernsey  

0.66 

0.71 

0  78 

Jersey. 

0  64 

0  61 

0  74 

0  83 

TABLE  XXII 
LACTOSE 


PROTEID 


RATIO 


Breed. 

Lythgoe. 

Eckles  and 
Shaw. 

New  Jersey 
Expt.  Stat. 

Holstein.     Frisian  
Dutch  belt 

1.60 
1  67 

1.54 

1.43 

Ayrshire 

1  63 

1  51 

1  39 

Shorthorn  

1  39 

1  47 

Guernsey. 

1  30 

1  22 

Jersey. 

1  43 

1  21 

1  22 

the  figures  are  considered  in  relation  to  the  fat  content  yielded 
by  each  breed,  it  will  be  found  that,  with  an  increasing  per- 


centage of  fat,  the 


proteid 


and 


lactose 


ratios   decrease   and 


fat  proteid 

that  the  percentage  of  fat  in  the  total  solids  increases. 

Seasonal  variations  are  also  shown  by  the  various  ratios  as 
will  be  seen  from  Table  XXIV,  the  figures  in  which  are  cal- 
culated from  those  in  Table  XIX. 


48  THE  NORMAL  COMPOSITION  OF  MILK 

TABLE  XXIII 
PERCENTAGE  OF  FAT  IN  TOTAL  SOLIDS 


Breed. 

Vieth. 

Lythgoe. 

Eckles  and 
Shaw. 

American 
Expt.  Stat. 

Jersey  

38.0 

38  3 

39  2 

34  Q 

Guernsey 

35  9 

Q4    Q 

Welsh  

34.7 

Sussex 

34  4 

Kerry. 

34.5 

Dairy  shorthorn  

31.3 

Pedigree  shorthorn  
Shorthorn 

31.4 

29  4 

2Q  3 

Red  polled  

32.8 

Ayrshire  

31  8 

29  6 

28  7 

Dutch  belt 

30  9 

Montgomery  

28.5 

Holstein.            

29  2 

27  2 

28  1 

TABLE  XXIV 
SEASONAL  VARIATIONS  IN  PROPORTIONS  OF  CONSTITUENTS 


Month 

Proteid 

Lactose 

Percentage  of  Fat 

Fat 

Proteid 

in  Total  Solids. 

January                 

0.94 

1  29 

29  8 

February 

0  95 

1  33 

29  2 

March                 

0.95 

1.37 

28  8 

April 

0  95 

1  33 

29  1 

May                        

1.00" 

1  31 

28  4 

June 

0  99 

1  34 

28  1 

July 

0.90 

1  42 

29  3 

August 

0  87 

1  41 

30  3 

September  

0.84 

1  42 

31  0 

October                       .    .    . 

0  86 

1  37 

30  9 

November 

0  87 

1  35 

30  9 

December  

0  92 

1  30 

30  2 

NON-FATTY  SOLIDS 


49 


The  influence  of  the  stage  of  lactation  upon  the  various  ratios 
is  shown  in  Table  XXV,  which  is  based  on  the  results  recorded 
in  Table  XVII. 


TABLE  XXV 

INFLUENCE  OF  STAGE  OF  LACTATION  ON  PROPORTIONS  OF 
CONSTITUENTS 

By  Four-week  Periods 


Period  No. 

Proteid 

Lactose 

Percentage  of  Fat 
in  Total  Solids. 

Fat    ' 

Proteid' 

1 

0.67 

1.47 

31.4 

2 

0.61 

1.58 

31.4 

3 

0.66 

1.62 

30.8 

4 

0.66 

1.55 

30.8 

5 

0.69 

1.50 

30.9 

6 

0.71 

1.46 

30.3 

7 

0.70 

1.50 

30.4 

8 

0.71 

1.46 

30.3 

9 

0.72 

1.32 

31.1 

10 

0.74 

1.22 

31.2 

11 

0.76 

1.23 

31.4 

12 

0.74 

1.22 

32.3 

13 

0.78 

1.13 

32.8 

14 

0.73 

1.23 

33.2 

The  above  results  show  that  the  general  tendency  during 
the  period  of  lactation  is  for  the  proteid  to  increase  with  the 

fat,  though  at  a  slightly  higher  rate.     This  increased      , 

-  iat 

ratio  with  increase  of  fat  percentage,  however,  is  not  capable 
of  general  application  as  the  results  show  that  the  reverse  is 
the  case  when  the  increase  in  fat  is  due  to  the  breed  of  the  cow. 
The  lactose  content  being  comparatively  constant,  its  ratio  to 
that  of  the  proteid  is  reduced  with  increase  of  percentage  of 
fat  owing  to  the  increased  proteid  content.  The  percentage 
of  fat  in  the  total  solids  increases  with  the  fat  as  the  extra  incre- 


50 


THE  NORMAL  COMPOSITION  OF  MILK 


ment  of  proteid  is  more  than  counterbalanced  by  the  constancy 
in  the  lactose  and  mineral  matter. 

The  percentage  of  ash  in  milk  is  comparatively  constant  but 
small  variations  are  observable  and  depend  upon  variations  in 
the  proteid  content,  as  a  portion  of  the  ash  is  combined  with  the 
caseinogen  to  form  the  caseinogen  complex.  Richmond  has 
deduced  the  formula  .4=0.36  +0.1  IP,  in  which  A  and  P  rep- 
resent the  percentages  of  ash  and  proteid,  for  the  calculation  of 
the  ash  content. 

It  is  upon  the  above  basic  relations  between  the  amounts  of 
the  various  constituents  in  milk  that  the  formulae  of  Van  Slyke, 

rp     o 

previously  referred  to,  and  that  of  Olsen  13,  P=T.  S.  —  r1^-' 

are  based.  Lythgoe  has  suggested  that  lactose  may  be  cal- 
culated from  the  following  formulae. 


from  Olsen's  formula  and 

L  =  T.  S.-[F+0.7+  (0.4(^-3)}  +2.8], 

from  Van  Slyke's  formula.  The  ash  in  these  formulae  is 
assumed  to  be  0.70  per  cent,  but  it  would  be  preferable  to  sub- 
stitute Richmond's  formula  of  A  =0.36  +0.1  IP  for  the  assumed 
value. 

All  the  foregoing  refers  only  to  whole  milk,  that  is,  the  mixed 
milk  obtained  by  continuous  milking  until  the  udders  are  dry. 
The  variations  due  to  partial  milking  are  very  striking  and 

TABLE  XXVI 

(BOUSSINGAULT) 


Portion. 

1 

2 

3 

4 

5 

6 

Total  solids  
Fat. 

10.47 
1  70 

10.75 
1  76 

10.85 
2  10 

11.23 
2  54 

11.63 
3  14 

12.67 
4  08 

Solids-not-fat  

8  77 

8  99 

8  75 

8  69 

8  45 

8  59 

NON-FATTY  SOLIDS 


51 


may  be  much  greater  than  those  caused  by  the  various  factors 
previously  discussed.  Analyses  showing  the  composition  of 
milk  obtained  at  various  stages  are  given  in  Tables  XXVI, 
XXVII  and  XXVIII. 

TABLE  XXVII 
AYRSHIRES  (AUTHOR) 


Fore  Milk. 

Middle  Milk. 

Strippings. 

Fat 

1  40 

5  90 

9  go 

Lactose           

4  95 

4  94 

4  87 

Proteid 

3  17 

2  98 

2  78 

Ash.  .               

0.80 

0.74 

0  71 

Lactose 

RatlOS    :R  r-r  .  . 

1  57 

1.66 

1  75 

Proteid 
Proteid 

0    0« 

OKI 

OOQ 

Fat     

TABLE  XXVIII 
AVERAGE  OF  JERSEYS,  SHORTHORNS,  AND  HOLSTEINS 

(ECKLES   AND 


Total 

Relative  Size 

Fat. 

Lactose. 

Proteid. 

Ash. 

Solids. 

of  Fat 
Globules. 

Fore  milk.  .  . 

1.87 

5.30 

3.58 

0.75 

10.47 

139 

Stoppings.  . 

6.28 

5.33 

3.38 

0.70 

14.86 

215 

The  physical  and  chemical  characteristics  of  the  butter  fat 
as  determined  by  Eckles  and  Shaw  were  as  follows : 

TABLE  XXIX 


Reichert- 
Meissl 

Iodine 

Saponification 

Melting 

Yellow 

Number. 

Number. 

Number. 

point. 

Colour. 

Fore  milk.  .  . 

27.2 

34.1 

230.1 

33.9 

39 

Strippings.  . 

26.3 

33.8 

228.3 

33.9 

39 

52  THE  NORMAL  COMPOSITION  OF  MILK 

All  these  results  show  that  the  chief  variation  in  the  com- 
position of  milk  at  various  stages  of  milking  is  due  to  fat,  and 
that  the  relative  proportions  of  the  plasma  constituents  remain 
comparatively  constant.  The  proteid  is  the  most  variable 
component  of  the  plasma  and  this  fact  is  reflected  in  the  in- 
creasing — — -rr  ratio  and  the  decreasing  ash  percentage.  The 

— - —  -  ratio  is  entirely  different  to  those  previously  stated  and 
i  a  t 

shows  the  entire  lack  of  organic  relation  between  these  two 
constituents.  This  points  to  the  variation  in  the  fat  content 
being  due  to  mechanical  causes  and  not  to  changes  in  meta- 
bolism. This  is  also  the  view  of  Kirchner,14  who  considered 
that  the  fat  globules  are  mechanically  retained  in  the  fine 
ducts  of  the  udder  and  escape  in  the  strippings.  Eckles  and 
Shaw  point  out,  in  support  of  this,  that  the  larger  the  pro- 
duction of  milk  the  greater  the  increase  in  fat  as  the  milking 
proceeds;  which  is  explained  by  the  hypothesis  that,  in  the 
heavier  milking  cows,  the  udder  is  more  congested  and  the 
openings  of  the  ducts  reduced  by  compression.  The  relative 
size  of  the  fat  globules  at  various  stages  of  milking  also 
supports  this  view. 

Colostrum.  The  name  "  colostrum  "  is  applied  to  the  udder 
secretion  before,  and  immediately  after,  parturition.  A  yellow 
viscous  secretion,  not  unlike  that  produced  by  pathological 
conditions,  is  often  formed,  but  this  is  replaced  several  days 
before  parturition  by  the  colostrum  proper.  Colostrum  is  a 
yellowish,  sometimes  reddish  (due  to  the  presence  of  blood), 
slimy  liquid  with  an  acid  reaction  and  which  shows  a  tendency 
to  separate.  Compared  with  ripe  milk  the  quantity  of  proteids 
in  colostrum  is  very  high  and  is  due  more  to  increases  in  albumin 
nuclein,  and  globulin  than  to  an  excess  of  caseinogen.  This 
points  to  glandular  inflammation  as  a  result  of  physiological 
irritation.  Cholesterol,  lecethin,  creatinine,  tyrosine,  and  urea 
are  also  present.  Dextrose  is  present  in  addition  to  lactose, 
which  is  slightly  diminished  in  quantity,  and  the  ash  is  higher 


COLOSTRUM 


53 


than  in  normal  milk.     The  microscopical  appearance  of  colos- 
trum is  characterised  by  the  presence  of  glandular  epithelium 

TABLE  XXX 
COMPOSITION  OF  COLOSTRUM  (SOTHURST) 


Milking 
Number. 

Total 
Solids 

Ash. 

Fat. 

Sugar. 

Total 
Proteid. 

Casein- 
ogen. 

Globulin. 

Albumin. 

1 

22.87 

1.03 

2.30 

2.74 

12.23 

4.86 

5.32 

1.45 

2 

16.23 

0.87 

2.49 

2.85 

6.97 

3.35 

2.04 

1.01 

3 

15.16 

0.86 

3.41 

3.37 

5.82 

3.09 

1.45 

0.75 

4 

.15.19 

0.82 

4.74 

3.62 

4.69 

2.70 

0.66 

0.78 

5 

15.74 

0.82 

5.10 

3.63 

4.01 

2.61 

0.55 

0.52 

6 

15.75 

0.82 

4.55 

3.86 

4.04 

2.56 

0.48 

0.49 

7 

15.72 

0.80 

5.49 

3.92 

3.46 

2.21 

0.31 

0.62 

8 

15.62 

0.80 

5.47 

4.57 

3.36 

2.17 

0.27 

0.61 

9 

15.47 

0.82 

5.62 

4.22 

3.35 

2.15 

0.25 

0.59 

11 

15.97 

0.84 

5.04 

3.82 

3.52 

2.52 

0.22 

0.59 

14 

16.55 

0.84 

5.15 

5.00 

3.21 

2.20 

0.20 

0.56 

16 

16.28 

0.83 

4.90 

5.01 

3.32 

2.34 

0.19 

0.55 

17 

16.06 

0.81 

4.79 

4.87 

3.24 

2.25 

0.19 

0.56 

TABLE  XXXI 
COMPOSITION  OF  COLOSTRUM  (ENGLING) 


./• 

Immediately 
After 
Calving. 

After 
10  Hours. 

After 
24  Hours. 

After 
48  Hours. 

After 
72  Hours. 

Specific  gravity  
Total  solids  
Caseinogen  

1.068 
26.83 
2.65 

1.046 
21.23 

4.28 

1.043 

19.37 
4.50 

1.042 
14.19 
3.25 

0.035 
13.36 
3.33 

Albumin  and  globulin  . 
Fat                        .  . 

16.56 
3  53 

9.32 
4.66 

6.25 
4.75 

2.31 
4.21 

1.03 
4.80 

Lactose 

3  00 

1  42 

2  85 

3  46 

4  10 

Ash  

1.18 

1.55 

1.02 

0.96 

0.82 

in  the  form  of  foam  cells  and  signet  ring-shaped  cells  with  so- 
called  moons  and  caps,  and  in  albuminophores.     Numerous 


54  THE  NORMAL  COMPOSITION  OF  MILK 

leucocytes  are  present  and  also,  during  the  first  few  days,  large 
numbers  of  erythrocytes. 

The  composition  of  colostrum  is  shown  in  Tables  XXX  and 
XXXI. 

According  to  Jensen  the  amylase  and  catalase  content  is 
increased  during  the  colostral  period  but  reductase  is  absent. 

ABNORMAL  AND  ADULTERATED  MILK 

Influence  of  Disease.  Although  the  chemical  examination 
of  milk  produced  under  pathological  conditions  is  of  but  little 
practical  importance  owing  to  the  infrequency  with  which  such 
conditions  exist  and  the  improbability  of  this  milk  being  sold 
unmixed  with  normal  milk,  it  is,  nevertheless,  of  interest  to 
consider  the  general  changes  that  occur.  Acute  diseases  asso- 
ciated with  great  pain  and  fever  are  usually  characterised  by  a 
rapid  diminution  in  the  quantity  of  milk  secreted.  In  general 
and  specific  infections  the  fat  may  be  either  increased  or  de- 
creased with  similar  fluctuations  in  the  ash  and  lactose  contents. 
According  to  Schnorf ,  most  of  the  internal  infections,  even  when 
the  udder  is  not  involved,  produce  a  diminution  in  the  lactose 
and  proteid  content  as  a  result  of  increased  metabolism.  Cata- 
lase, especially  in  peritonitis  and  tuberculosis,  may  be  consid- 
erably increased  and  changes  in  taste  and  coagulability  may 
result  from  general  infections. 

Although  it  is  well  known  that  the  composition  of  milk 
changes  with  alterations  in  the  function  and  condition  of  the 
secreting  organs,  comparatively  little  is  known  regarding  the 
influence  of  diseases  of  the  udder  upon  the  various  constituents 
of  the  milk.  Many  analyses  have  been  made  and  various  ob- 
servers have  obtained  what  are  apparently  discordant  results, 
but  this  may  be  attributed  to  factors  such  as  intensity  and 
duration  of  the  disease  being  different. 

In  acute  forms  of  mastitis,  caused  by  organisms  of  the  colon 
group,  or  streptococci,  or  in  mixed  infections,  the  milk  may 
have  a  bloody  discolouration,  Liter  becoming  more  like  colos- 


INFLUENCE  OF  DISEASE 


55 


trum  in  appearance  and  finally  changing  to  a  thick  yellowish 
secretion  containing  many  dark  flakes  in  a  clear  serum. 

In  chronic  infections  the  changes  are  gradual  and  the  ap- 
pearance and  composition  of  the  milk  may  be  almost  normal 
for  a  time ;  sooner  or  later,  however,  the  cell  content  is  increased 
with  a  consequent  increase  in  the  albumin,  and  erythrocytes 
cause  a  discolouration  of  the  sediment  on  standing. 

In  udder  infections  the  fat  usually  decreases  but  may  fluc- 
tuate rapidly  within  rather  wide  limits;  the  lactose  and  casein- 
ogen  usually  decrease  slightly,  but  the  decrease  in  the  latter 
constituent  is  more  than  counterbalanced  by  a  marked  increase 
in  the  albumin,  resulting  in  an  abnormally  high  proteid  content. 

m  ,,    .  IT    proteid        ,    lactose 

These  changes  result  in  very  abnormal       ,  ,       and  — ^ 

fat  proteid 

ratios  as  is  shown  in  Table  XXXII.  On  account  of  the  bac- 
terial origin  of  these  infections,  the  enzymes  in  the  milk  are  very 
much  increased. 

TABLE  XXXII 
EFFECT  OF  DISEASE  ON  COMPOSITION  OF  MILK 

(SCHAFFER  AND  BENDZYNSKl) 


Total 

Fat 

Ash 

Proteid 

Lactose 

Solids. 

Fat 

Proteid' 

Non  infectious  garget  .  .  . 
Yellow  garget. 

7.17 
10  66 

0.82 
1  99 

0.53 
1  84 

4.01 
6  00 

0.79 
0  83 

4.89 

3  01 

0.13 
0  31 

Parenchymatous  mastitis 

9.74 

2.16 

1.01 

4.21 

0.99 

1.99 

0.24 

Another  cause  of  abnormal  composition  of  milk  is  the  cessa- 
tion of  the  lactation  period.  This  has  already  been  discussed 
on  page  49  where  it  was  shown  that  during  the  last  stages  of 


lactation,  the 


ratio  decreased  considerably  owing  to  the 


proteid 
increased  proteid  percentage. 

Milk  Adulteration.    Artificial  abnormalities  in  the  com- 
position of  milk  produced  by  the  addition  of  extraneous  sub- 


56  THE  NORMAL  COMPOSITION  OF  MILK 

stances  or  by  the  abstraction  of  the  natural  constituents,  gen- 
erally by  human  agency,  is  usually  conveyed  by  the  term  milk 
adulteration,  and  this,  strictly  speaking,  has  no  reference  to 
any  standard  that  may  be  adopted. 

For  the  detection  of  adulteration,  a  complete  determination 
of  the  various  constituents  of  the  sample  should  be  made  and  the 
amounts  of  fat,  lactose,  proteid,  and  ash  so  found  compared 
with  the  percentages  as  calculated  from  the  formula  of  Van 

j  ™-  i,        j      rm.    proteid       ,   lactose 

Slyke,  Olsen,  and  Richmond.     The      £  .  -  and  —  — —  ratios 

fat  proteid 

should  also  be  calculated.  The  addition  of  water  does  not 
give  proteid  values  which  are  materially  different  from  those 
calculated  by  the  Olsen  formula  but  are  invariably  less  than 
those  calculated  by  the  Van  Slyke  formula,  the  difference  being 
proportional  to  the  amount  of  water  added.  The  PVS  (proteid 
calculated  by  the  Van  Slyke  method)  in  this  case,  is  greater 
than  the  P.  0.  (proteid  calculated  by  the  Olsen  method).  The 

,,.,.         r  ,,      proteid        ,    lactose      ,. 

addition  of  water  leaves  the       ,  .  -  and  — —^  ratios  un- 

fat  proteid 

changed. 

The  amount  of  proteid  found  by  direct  estimation  in  the 
case  of  abstraction  of  fat  would  be  greater  than  either  of  the 
calculated  values,  and  in  this  case  P.O.  would  be  greater  than 
PVS.  This  is  due  to  the  Van  Slyke  formula  being  based  on  the 

constituent   which   has   been   abstracted.     The   — — -^  ratio 

proteid 

would  be  normal  and  the  pr°  ^     ratio  abnormally  high.     In 

lat 

both  of  these  instances  the r-:  ratio  is  unaltered  and  this 

proteid 

is  valuable  in  distinguishing  between  naturally  abnormal  milks 
and  those  rendered  abnormal  by  external  agencies.  High 

r-:-  ratios  are  extremely  rare  but  low  ones  may  be  pro- 

proteid 

duced  by  the  various  causes  previously  mentioned. 

The  refractive  power  of  the  serum  should  also  be  considered 


MILK  ADULTERATION  57 

in  connection  with  samples  suspected  of  being  adulterated. 
The  index  of  refraction  is  reduced  by  the  addition  of  water  but 
is  unaltered  by  fat  abstraction.  The  following  are  the  mini- 
mum figures  for  genuine  milks  when  prepared  by  the  usual 
methods. 

TABLE  XXXIII 
REFRACTOMETER  VALUES  FOR  MILK  SERUM 


Method  of  Preparation  of  Serum. 

Reading  on  Zeiss  Immersion 
Refractometer. 

Copper  sulphate  

36 

Acetic  acid 

40 

Natural  souring 

38 

Although  the  above  methods  are  capable  of  detecting  the 
abstraction  of  small  quantities  of  fat,  their  possibilities  regarding 
the  indication  of  added  water  are  more  limited,  and  it  is  doubtful 
if  they  could  be  relied  upon  to  detect  additions  smaller  than 
would  be  necessary  to  reduce  the  total  solids  or  solids-not-fat 
below  the  requirements  of  any  reasonably  high  standard. 
Even  though  these  methods  are  reliable  for  the  detection  of  the 
abstraction  of  small  amounts  of  fat,  the  advisability  of  using 
them  as  a  basis  for  the  certification  of  adulteration,  when  the 
fat  exceeds  the  standard,  is  extremely  doubtful  owing  to  the 
difficulty  of  securing  a  conviction.  Those  whose  duties  embrace 
the  analysis  of  public  milk  supplies  meet  many  of  these  examples 
and  have,  unfortunately,  no  option  but  to  report  them  as  gen- 
uine, although  they  are  undoubtedly  sophisticated.  This  is 
one  of  the  inherent  disadvantages  of  minimum  standards. 

The  addition  of  cane  sugar  or  dextrin  to  watered  milk  for 
the  purpose  of  increasing  the  non-fatty  solids  is  indicated  by  a 

low  proteid  value,  an  abnormally  high  -       4-r  ratio,  and  a 

deficiency  of  ash.     Methods  for  the  detection  and  estimation 
of  cane  sugar  are  given  on    page    88.     Glycerine  and  starch 


58  THE  NORMAL  COMPOSITION  OF  MILK 

have  also  been  employed  as  counterfeits  for  non-fatty  solids 
reduced  by  the  addition  of  water. 

CALCULATION  OP  ADULTERATION 

Added  Water.    The  probable  amount  of  water  added  to 
milk  may  be  calculated  from  the  formula 

Added  water  =  100  --  ^-XlOO  in  which  SNF  represents 
snf 

the  amount  of  solids-not-fat  found,  and  snf  the  average  amount 
of  solids-not-fat  found  in  genuine  milks  during  the  same  season. 
If  such  records  are  not  available  a  value  of  8.8  may  be  assumed. 
Where  minimum  standards  are  in  force  the  value  in  the  standard 
is  substituted  in  the  above  formula,  whether  it  be  for  solids- 
not-fat  or  total  solids.  Thus 

SNF  found 

Added  water  =  100  --  —  --  7—  r:  -  -=  X  100, 
minimum  snf  allowed 

1ftn  __  T.S.  found 

minimum  T7.  fallowed  X1C 

The  added  water  calculated  by  this  latter  method  is  usually 
stated  in  the  certificate  of  analysis  as  "at  least  .  .  .  per  cent." 
Another  formula  for  calculating  the  added  water  is 

G+F 

Added  water  =  100  —  TT  *  ^  where  G  =  degrees  of  gravity 


or  lactometer  reading,  and  F  =  the  percentage  of  fat.  The  prob- 
able amount  added  may  be  obtained  by  substituting  36.0  for 
34.5. 

Fat  Abstraction.  The  removal  of  cream  is  indicated  by 
an  abnormally  low  fat  content  and  the  minimum  amount  of 
fat  abstraction  may  be  calculated  from  the  formula. 

Fat  abstracted  =  100-  4x100   where   /,    and   F,  are   the 
r 

amounts  of  fat  found  in  the  sample  and  the  minimum  required 
by  the  standard,  respectively.  The  probable  amount  removed 
may  be  obtained  by  substituting  the  average  value  for  the 
month  in  which  the  sample  is  taken. 


MILK  STANDARDS  59 

MILK  STANDARDS 

For  the  regulation  of  the  sale  of  milk,  various  standards 
have  been  established  which  the  mixed  milk  of  a  herd  of  cows 
might  reasonably  comply  with,  and  it  is,  at  least,  this  mini- 
mum quality  that  a  purchaser  expects  to  be  supplied  with.  In 
England  no  specific  standard  has  been  adopted  by  statute  but  a 
standard  of  3.0  per  cent  of  fat  and  8.5  per  cent  of  solids-not-fat 
was  adopted  many  years  ago  by  the  Society  of  Public  Analysts 
as  a  guidance  for  analysts  for  milk  that  is  of  the  nature,  sub- 
stance, and  quality  that  might  reasonably  be  demanded  by  the 
purchaser.  The  onus  of  proof  regarding  this  contention,  how- 
ever, was  upon  the  analyst,  and  it  was  not  until  1901  that  this 
was  transferred  to  the  vendor  by  an  order  of  the  Board  of  Agri- 
culture which  stated  that  milk  containing  less  than  3.0  per  cent 
of  fat  or  8.5  per  cent  of  solids-not-fat  shall  be  presumed  to  be 
not  genuine  until  the  contrary  is  proved.  This  has  led  to  the 
"  appeal  to  the  cow  "  or  the  "  stall  "  or  "  byre  "  test  in  which 
the  cows  are  completely  milked  in  the  presence  of  a  witness 
or  witnesses  and  the  milk  afterwards  analysed  for  comparison 
with  the  previous  sample.  If  the  results  agree,  the  sample  is 
to  be  regarded  as  genuine  and  to  comply  with  the  provisions  of 
the  Food  and  Drugs  Act.  It  is  obvious  that  great  care  should 
be  taken  in  obtaining  the  test  sample  by  insisting  upon  all  the 
cows  being  thoroughly  stripped  of  milk  and,  if  possible,  making 
the  test  on  the  same  day  of  the  week  and  at  the  same  milking  from 
which  the  first  sample  was  obtained.  Such  a  procedure  evidently 
regards  milk  as  the  secretion  of  healthy  cows  without  having 
regard  to  the  breed,  nature  and  quantity  of  food  supply,  and 
treatment  of  the  cow,  and  this  is  apparently  also  the  view  of  the 
Scottish  High  Court  of  Judiciary  as  expressed  during  the  appeal 
of  Scott  v.  Jack.  Lord  Johnston  expressed  the  opinion  that 
"  milk  in  the  sense  of  the  statute  is  milk  drawn  from  the  cow, 
not  milk  in  the  process  of  formation  in  the  chyle,  in  the  blood, 
in  the  glands  of  the  cow.  ..."  This  decision  that  milk  is  to  be 
regarded  as  the  secretion  of  healthy  cattle  leaves  much  to  be 


60  THE  NORMAL  COMPOSITION  OF  MILK 

desired,  as  any  breed  may  be  used  and  the  ration  adjusted  to 
secure  quantity  rather  than  quality  and  so  lead  to  a  diminution 
of  both  the  average  and  minimum  composition  of  the  normal 
secretion. 

The  breeding  of  dairy  cattle  on  scientific  principles  has  led 
to  the  introduction  of  strains  which  secrete  large  quantities  of 
milk  of  comparatively  poor  quality;  the  total  weight  of  butter 
fat  produced  is  at  a  maximum  and  when  such  milk  is  to  be  used 
for  butter  making  this  method  of  breeding  must  be  regarded  as 
legitimate  and  commended  as  a  step  forward  in  intensive 
breeding.  When  such  produce  is  intended  for  sale  as  milk 
a  very  different  view  must  be  taken  of  such  methods  for,  as 
regards  the  ultimate  effect,  there  is  no  difference  .between 
this  process  and  the  deliberate  addition  of  water  to  milk  of 
superior  quality.  If  milk  is  to  be  regarded  as  the  secretion  of 
cows,  without  additions  or  abstraction,  it  is  evident  that  a 
premium  is  placed  upon  quantity  regardless  of  quality,  with  the 
consequence  that  the  water  content  of  milk  will  become  in- 
creasingly greater.  It  might  be  argued  that  such  a  course  of 
reasoning  is  merely  hypothetical  inasmuch  as  the  average 
composition  of  milk  shows  no  definite  tendency  to  deteriorate 
from  decade  to  decade.  Unfortunately  there  are  compara- 
tively few  reliable  records  of  data  covering  considerable  periods. 
The  records  of  the  Aylesbury  Dairy  Co.,  London,  as  published 
by  Droop  Richmond,  show  that  the  milk  supplied  in  1912  was 
but  very  little  different  in  composition  to  that  supplied  in  1900. 
The  intervening  period  is  marked  by  a  rise  in  quality  in  1902  and 
1903  after  which  there  is  a  steady  decline.  The  results  are  set 
out  in  Diagram  II. 

The  conditions  in  New  York  City  present  an  entirely  differ; 
ent  aspect  of  this  question.  Prior  to  1910  the  standard  de- 
manded at  least  12  per  cent  of  total  solids,  but  in  that  year  the 
interests  representing  the  Holstein  breeders  were  strong  enough 
to  effect  a  reduction  of  the  standard  to  11.5  per  cent.  When 
this  new  standard  became  operative,  no  "  quid  pro  quo  "  in 
the  shape  of  a  reduction  in  price  was  received  by  the  consumer, 


MILK  STANDARDS 


61 


although  the  report  of  the  Health  Department  states  that 
"  the  reduction  is  a  stimulus  to  adulteration  and  that  the 
records  of  the  department  show  that  certain  dealers,  who, 
under  the  old  law,  were  just  within  the  standard  of  12  per  cent, 
are  now  selling  milk,  which  repeated  analyses  have  shown  to  be 
just  within  the  lowered  standard  of  11.5  per  cent  of  total  solids." 
In  this  case  it  is  evident  that  the  quality  of  the  milk  supplied, 
by  at  least  a  portion  of  the  producers,  followed  the  standard, 


DIAGRAM  II 

YEARLY  VARIATION  IN  COMPOSITION  OF  MILK  (DROOP  RICHMOND) 


Percentage  of  Fat 

w  eo  co  «» 

W  05  -J  00 

^* 

V 

to  S3  .  5  to 

W  0»  '_  00 

Percentage  of  Total  Solids 

A 

t 

S 

/  ) 

\    ^ 
\ 
\ 
\ 

X 

x^^ 

/. 

X 

i^^ 

A 

1 
1 

5 

>***** 

\ 

\ 

/ 

\ 

<•** 

/ 

J 

\ 

Nj 

/ 

i 
\ 

Fat 

\ 

S 

Total  Solids. 

1900  1901   1902   1903   1904  1905   1906  19»7  1908   1909   1910  1911  .1912  - 


and  it  seems  inevitable  that  the  other  producers  will  be  driven 
to  the  adoption  of  similar  measures  by  stress  of  competition. 

In  both  the  United  States  and  Canada,  milk  standards  are 
of  an  entirely  different  legal  nature  to  those  obtaining  in  Great 
Britain;  the  minimum  limits  of  composition  are  clearly  defined 
by  ordinance  or  statute  and  admit  of  no  appeal  to  the  cow. 
These  standards  are  to  be  regarded  as  specifications  of  what  is 
required  to  be  sold  as  milk  and  not  the  minimum  quality  that 
might  reasonably  be  expected  by  the  purchaser.  This  is 
equitable,  as  the  purchaser,  for  a  given  price,  should  receive 


62  THE  NORMAL  COMPOSITION  OF  MILK 

an  article  of  definite  quality  and  not  something  that  may  be  the 
minimum  quality  produced  by  natural  variations.  To  achieve 
this,  the  dairyman  must  so  grade  his  herd  that  the  mixed  milk 
will  at  all  times  comply  with  the  standard.  It  may  be  argued 
that  a  rigid  interpretation  of  a  standard  may  inflict  unnecessary 
hardship  on  producers  by  reducing  what  is  usually  but  a  com- 
paratively small  margin  of  profit,  but  it  is  surely  preferable 
that  the  economic  balance  between  producer  and  consumer 
should  be  adjusted  by  an  increased  price  rather  than  by  a  deter- 
ioration in  quality.  The  adjustment  by  price  is  understood  by 
everyone  whereas  the  maintenance  of  the  balance  by  a  reduction 
in  quality  is  an  invidious  one  only  capable  of  being  correctly 
appreciated  by  experts. 

Rigid  enforcement  of  standards  is  also  necessary  in  the 
interests  of  dairymen  in  order  to  prevent  unfair  competition, 
as  it  is  obviously  unfair  to  allow  some  to  breed  for  quantity 
and  supply  a  quality  which  is,  perhaps,  only  occasionally  just 
below  the  standard,  whilst  others  are  supplying  milk  which  is 
invariably  above  the  standard.  One  typical  example  of  this 
unfair  competition  which  the  author  experienced  was  the  case 
of  producer  X,  who  kept  pure-bred  Holsteins,  which  produced 
milk  of  the  required  standard,  12  per  cent  of  total  solids  and 
3.0  per  cent  fat,  during  the  greater  part  of  the  year,  but  just 
failed  to  meet  it  during  the  season  when  the  cows  "  freshened." 
An  examination  of  the  herd  during  this  period  showed  that  nine 
cows,  out  of  the  22  head  comprising  the  herd,  secreted  a  low 
quality  of  milk  and  were  giving  an  abnormally  large  quantity, 
one  cow  producing  as  much  as  7J  gallons  per  day.  This  pro- 
ducer had  an  obvious  advantage  over  others  whose  herds  were 
graded  with  Ayrshires  and  other  breeds  giving  a  higher  quality 
but  a  smaller  quantity. 

The  standards  prescribed  in  various  countries  show  but 
small  differences;  those  prevailing  in  States,  provinces  and 
cities,  which  have  power  to  make  local  regulations  unfor- 
tunately show  larger  variations  and  these  often  conflict  with 
those  of  contiguous  authorities.  Table  XXXIV  gives  a  fairly 


MILK  STANDARDS 


63 


complete  list  of  the  standards  for  milk  and  cream  obtaining  in 
English-speaking  countries. 

TABLE  XXXIV 
MILK  AND  CREAM  STANDARDS 


Country,  State  or 

MILK. 

SKIM 
MILK. 

CREAM. 

Province. 

Total 
Solida. 

Fat. 

Solids- 
Not-fat. 

Solids- 
Not-fat. 

Fat. 

Great  Britain 

3  00 

8  50 

Australia. 
New  South  Wales. 

3   20 

8  50 

8  80 

"Full"           "Half" 
35  0          25  0 

South  Australia  .  . 
Victoria 

12.00 
12  00 

3.25 
3  50 

8.50 
8  50 

8.80 
8  80 

"Double"       "Single" 
35.0          25.0 

"a—'   "»" 
35  0          25  0 

Queensland 

12.00 

3.30 

8.50 

8  80 

Cream. 

35  0 

Western  Australia 

Tasmania  
Canada.  Dominion' 

11.70 
12.00 

3.20 

3.30 
3  25 

8.50 

8.50 
8.50 

8.80 

8.80 
8  50 

"Double"       "Single" 

35.0          25.0 

Cream. 

35.0 
18  0 

Alberta  
British  Columbia  . 
Manitoba 

12.00 
11.75 

3.00 
3.25 
3  25 

9.00 
8.50 
8  50 

8.50 
8  50 

18  0 

New  Brunswick  .  . 
Nova  Scotia  
Ontario  
Quebec 

No 
No 
12.00 
12  00 

Stand 
Stand 
3.00 
3  00 

ards 
ards 

9  00 

18.0 
16  0 

Saskatchewan 

12  00 

3  50 

20  0 

South  Africa. 

3.00 

8.50 

25  0 

New  Zealand  . 

3.25 

8.50 

8.80 

/   25.0 

India. 
Calcutta  
Bombay 

11.50 

12.00 

3.00 
3.50 

8.50 
8.50 

\   40.0 

64 


THE  NORMAL  COMPOSITION  OF  MILK 


TABLE  XXXIV—  (Continued) 
MILK  AND  CREAM  STANDARDS 


Country,  State  or 

MILK. 

SKIM 
MILK. 

CREAM. 

Province. 

Total 

Solids. 

Fat. 

Solids- 
Not-fat. 

Solids- 
Not-fat. 

Fat. 

United  States.   Federal. 

3  25 

8   50 

9  25 

18  0 

California  

3  00 

8  50 

8  80 

18  0 

Colorado  

3  00 

16  0 

Connecticut  
District  of  Columbia.  .  . 
Florida  

11.75 
12.50 

3.25 
3.50 
3  25 

8.50 
9.00 
8  50 

'Q.'SO' 

9  25 

16.0 
20.0 
18  0 

Georgia  

3.25 

8  50 

9  25 

18  0 

Idaho 

11  20 

3  00 

8  00 

9  30 

18  0 

Illinois 

3  00 

8  50 

9  25 

18  0 

Indiana 

3  25 

8  50 

9  25 

18  0 

Iowa. 

12  00 

3  00 

16  0 

Kansas. 

3  25 

18  0 

Kentucky 

3  25 

8  50 

9  25 

18  0 

Louisiana. 

75 

3  50 

8  50 

8  00 

Maine 

11  75 

3  25 

8  50 

18  0 

Maryland    .    ... 

12  50 

3  50 

9  25 

18  0 

Massachusetts 

12.15 

3  35 

9  30 

15  0 

Michigan  

12.50 

3  00 

18  0 

Minnesota  

13.00 

3.25 

9  75 

20  0 

Missouri  

12.00 

3.25 

8.75 

9.25 

18  0 

Montana  

11.75 

3.25 

8.50 

20  0 

Nebraska  

3.00 

18  0 

New  Hampshire  

12.00 

8.50 

18.0 

New  Jersey. 

11  50 

3  00 

9  25 

16  0 

Nevada 

11  75 

3  25 

8  50 

9  25 

18  0 

New  York. 

11  50 

3  00 

18  0 

North  Carolina. 

11  75 

3.25 

8.50 

9  25 

18  0 

North  Dakota 

12  00 

3.00 

15  0 

Ohio. 

12  00 

3.00 

Oregon. 

11.70 

3.20 

8.50 

18  0 

Pennsylvania 

12.00 

3.25 

8.50 

18  0 

Rhode  Island  
South  Dakota 

12.00 

3.50 
3  25 

8  50 

9  25 

18  0 

Tennessee          

12.00 

3.50 

8.50 

9.00 

20.0 

Texas             

12.00 

3.25 

8.50 

9.25 

18.0 

Utah     

12.00 

3.20 

8.50 

18.0 

Vermont    

11.75 

3.25 

8.50 

9.25 

18.0 

Virginia 

3.25 

8.50 

9  25 

18  0 

Washington              .    .  . 

12.00 

3.25 

8.75 

9.30 

18  0 

3  00 

8  50 

9  00 

18  0 

BIBLIOGRAPHY  65 


BIBLIOGRAPHY 

1.  Bunge.     Path,  and  Phys.  Chemistry.     2d  English  Edition  trans,  by 
Starling.     1902,  104-105. 

2.  Lythgoe.     Ind.  and  Eng.  Chem.     1914,  6,  901. 

3.  Eckles  and  Shaw.     Bull.  156  U.  S.  A.  Dept.  of  Agr. 

4.  Morgen  et  al.     Landw.  Versuch.  Stat.     1904,  61,  1-284,  ibid.,  1906, 
64,  93-242. 

5.  Malmdjac.     J.  Pharm.     1901,  vi,  14,  70-74. 

6.  Fleichmann.     Untersuchung  der  Milch  von  sechszehn  Kiihen.    Landw- 
schaftliche  Jahrbiicher.     Vol.  20,  sup.  2,  Berlin,  1891. 

7.  Richmond.     Dairy  Chemistry.     London,  1914. 

8.  Eckles  and  Shaw.     Bull.  157  U.  S.  A.  Dept.  of  Agr. 

9.  Eckles  and  Shaw.     Bull  155  U.  S.  A.  Dept.  of  Agr. 

10.  Richmond.     Analyst.  37,  300. 

11.  Richmond.     Dairy  Chemistry.     London,  1914,  p.  160. 

12.  Van  Slyke.     Jour.  Amer.  Chem.  Soc.,  30,  1166. 

13.  Olsen.     Ind.  and  Eng.  Chem.,  I,  256. 

14.  Kirchner.    Handbuch,  der  Milchwirtschaft.     1898,  58. 


CHAPTER  III 
CHEMICAL  EXAMINATION 

ALTHOUGH  the  extent  of  the  chemical  examination  of  milk 
required  in  public  health  work  is  usually  confined  to  the  deter- 
mination of  the  fat  and  total  solids  and  the  detection  of  pre- 
servatives, a  brief  description  of  reliable  methods  for  the  esti- 
mation of  other  constituents  will  also  be  given  in  this  chapter 
as  they  are  invaluable  for  the  correct  diagnosis  of  sophistication. 

As  the  great  majority  of  ordinances  and  statutes  regulating 
the  sale  of  milk  contain  no  reference  to  constituents  other  than 
fat  and  total  solids,  these  will  be  considered  first. 

Estimation  of  Fat.  The  various  methods  introduced  for 
the  determination  of  fat  in  milk  may  be  divided  into  three 
groups. 

(1)  Volumetric  estimation  of  the  fat  brought  to  the  surface 
by  centrifugal  force  after  liberation  by  the  addition  of  chemicals. 

(2)  Ethereal  extraction  of  the  fat  liberated  by  the  addition 
of  chemicals. 

(3)  Ethereal  extraction  of  the  dried  milk. 

The  methods  which  comprise  the  second  group,  though 
invaluable  for  dealing  with  milk  products,  are  not  in  general 
use  for  the  examination  of  fresh  milk  and  will  not  be  given  in 
detail. 

The  mechanical  methods  of  group  one  are  now  in  almost 
universal  use  and  are  capable,  in  practised  hands,  of  yielding 
accurate  results.  The  three  chief  mechanical  methods  are  the 
Leffmann-Beam,  Babcock,  and  Gerber.  In  England,  the  Leff- 
man-Beam  and  the  Gerber  are  almost  exclusively  used  whilst 
in  America,  although  both  the  Babcock  and  Gerber  processes 
are  official,  the  former  is  more  generally  employed. 

66 


GERBER  METHOD  67 

Leffmann-Beam  Process.  15  c.cms.  of  the  sample  are 
transferred  by  means  of  a  pipette  into  a  flat-bottomed  bottle 
provided  with  a  narrow  neck  graduated  into  80  divisions,  10  of 
which  correspond  to  1  per  cent  of  fat  by  weight.  9  c.cms.  of 
concentrated  commercial  sulphuric  acid  are  then  added  in  three 
portions  with  thorough  admixture  after  each,  and  finally, 
3  c.cms.  of  a  mixture  of  equal  volumes  of  concentrated  hydro- 
chloric acid  and  amyl  alcohol.  After  shaking,  the  bottle  is 
filled  to  the  zero  mark  with  hot  dilute  sulphuric  acid  (1  in  2) 
and  whirled  in  the  centrifuge  for  3  to  4  minutes.  The  fat  rises 
to  the  top  of  the  liquid  as  a  yellowish  coloured  layer  and  the 
percentage  is  read  off  by  deducting  the  reading  at  the  junction 
of  the  fat  and  acid  from  the  reading  at  the  extreme  top  of  the 
fat,  not  the  bottom  of  the  meniscus. 

Babcock  Method.  This  method  differs  from  the  Leffmann- 
Beam  process  in  but  a  few  details.  The  bottle  neck  is  divided 
into  50  divisions  each  representing  0.2  per  cent  of  fat  by  weight 
of  the  17.6  c.cms.  employed.  The  procedure  is  as  follows: 
the  milk  having  been  placed  in  the  bottle  17.5  c.cms.  of  com- 
mercial sulphuric  acid  are  gradually  added  with  constant  agi- 
tation until  the  caseinogen  is  dissolved.  The  bottle  is  then 
placed  in  a  centrifuge  and  whirled  for  four  minutes  at  600  to 
1200  revolutions  per  minute,  according  to  the  diameter  of  the 
machine;  hot  water  is  added  until  the  bottle  is  filled  to  the 
lower  end  of  the  neck,  whirled  for  one  minute,  then  filled  to  the 
zero  mark  with  hot  water  and  whirled  for  one  further  minute 
to  bring  the  fat  layer  into  the  graduated  neck.  The  per- 
centage of  fat  is  then  read  off  as  in  the  Leffman-Beam  method, 
care  being  taken  that  all  readings  are  made  between  130°  and 
150°  F.  when  the  fat  is  quite  liquid.  The  author  has  found  that 
the  indistinct  line  of  demarkation  between  the  fat  and  the  acid 
occasionally  found  with  this  process  can  be  obviated  by  the 
addition  of  1  c.cm.  of  amyl  alcohol  after  the  addition  of  the  acid. 

Gerber  Method.  This  differs  from  the  modified  Babcock 
described  only  in  the  size  and  type  of  bottle,  and  quantities  of 
acid  and  milk  employed.  11  c.cms.  of  milk,  1  c.cm.  of  amyl 


68  CHEMICAL  EXAMINATION 

alcohol,  and  10  c.cms.  of  sulphuric  acid  are  mixed  in  the  usual 
way,  rotated  for  three  minutes,  then  immersed  in  a  water  bath 
at  140°  F.  for  a  minute  and  the  percentage  of  fat  read  off  on  the 
graduated  neck. 

Skim  milk  is  treated  exactly  as  ordinary  milk  except  in  the 
Gerber  process  in  which  two  to  three  minutes  shaking  are 
required  previous  to  whirling  and  a  longer  period  is  given  in  the 
water  bath  to  bring  the  temperature  to  140°  F. 

For  cream,  special  bottles  are  provided  in  the  Babcock 
method,  but  the  ordinary  ones  may  be  used,  as  in  the  Leffmann- 
Beam  method,  with  a  reduced  quantity  of  sample.  An  appro- 
priate weight  of  the  sample  is  washed  into  the  bottle  with  suf- 
ficient water  to  bring  the  total  volume  to  the  normal  volume  of 
the  bottle,  and  the  determination  carried  out  as  in  the  case  of 
milk.  The  result  is  multiplied  by  the  ratio  of  the  normal 
weight  of  the  method  (Leffmann-Beam  15.5  grms.,  Babcock 
18.0  grms.)  to  the  weight  of  the  sample  taken.  In  the  Gerber 
process  (normal  weight  11.35  grms)  0.5  gram  of  cream,  6  c.cms. 
of  hot  water,  1  c.cm.  of  amyl  alcohol,  and  6.5  c.cms.  of  acid 
are  used  with  a  further  addition  of  6  c.cms.  of  hot  water  pre- 
vious to  rotation. 

GRAVIMETRIC  METHODS 

Gottlieb's  Method.  In  this  method,  which  is  probably 
the  best  known  one  of  group  two,  the  caseinogen  is  dissolved  in 
ammonia  and  the  liquid  then  extracted  with  ether  and  petro- 
leum ether.  The  solution  of  fat  is  evaporated  and  the  residue 
weighed.  For  further  details  of  this  process  Richmond's 
Dairy  Chemistry  (Chas.  Griffin  &  Co.,  London,  1914)  should 
be  consulted. 

Adam's  Method.  5  grams  of  milk  are  weighed  out  in  a 
porcelain  or  glass  dish  and  absorbed  on  a  coil  of  fat  free  paper 
(special  strips  of  fat-free  paper  are  manufactured  for  this  pur- 
pose by  various  firms).  The  dish  and  coil  are  placed  in  the 
water  oven  until  thoroughly  dry  when  the  coil  is  placed  in  a 
Sohxlet  extraction  cone  and  the  residue  in  the  dish  extracted 


SPECIFIC  GRAVITY  69 

several  times  with  absolute  ether.  The  ether  so  used  is  poured 
over  the  coil  and  cone,  previously  placed  in  the  extraction 
apparatus,  and,  after  the  volume  of  solvent  has  been  increased, 
the  apparatus  is  connected  with  a  condenser  and  heated  in  a 
water  bath  at  about  45°  C.  After  four  or  five  hours  extraction 
the  ether  is  distilled  off  and  the  fat  dried  to  constant  weight. 
The  removal  of  the  ether  is  facilitated  by  drawing  a  current  of 
air  through  the  flask  by  means  of  a  vacuum  pump.  It  is  nec- 
essary that  the  ether  used  in  this  process  should  be  perfectly 
dry,  as  otherwise  small  quantities  of  milk  sugar  and  salts  are 
extracted  with  the  fat. 

This  is  the  official  method  of  the  Society  of  Public  Analysts 
of  Great  Britain  and  one  of  the  official  methods  of  the  Amer- 
ican Official  Association  of  Agricultural  Chemists. 

Total  Solids.  These  may  be  determined  either  directly 
by  drying  to  constant  weight  or  indirectly  by  calculation  from 
the  fat  content  and  the  specific  gravity. 

Direct  Method.  Five  grams  of  milk  are  weighed  into  a 
shallow  platinum  or  quartz  dish  and  after  all  visible  liquid  has 
been  driven  off  on  the  water  bath,  the  dish  and  contents  are 
dried  to  constant  weight  in  a  steam  oven.  Ignited  sand  or 
asbestos  may  be  used  to  facilitate  the  drying  process. 

Ash.  The  residue  from  the  determination  of  the  total 
solids  may  be  ignited  at  a  low  temperature  until  white  and  the 
residue  weighed,  or  a  fresh  portion  of  20  c.cms.  evaporated 
with  the  addition  of  6  c.cms.  of  nitric  acid,  and  ignited  until 
free  from  carbon  at  a  temperature  just  below  redness.  The 
former  method  is  the  more  convenient  and  the  latter  the  more 
accurate  one. 

Specific  Gravity.  This  is  determined  either  by  a  lac- 
tometer, a  Westphal  balance,  or  the  ordinary  specific  gravity 
bottle.  The  lactometer  method  is  the  simplest  and  quickest, 
but,  owing  to  the  comparatively  short  space  occupied  by  each 
graduation  (usually  1°)  and  the  opalescence  of  the  liquid  the 
degree  of  accuracy  obtained  is  low. 

The  gravity  is  usually  expressed  as  the  excess  weight  of 


70  CHEMICAL  EXAMINATION 

1000  c.cms.  of  milk  at  60°  F.  over  an  equal  volume  of  water  at 
the  same  temperature.  Thus,  a  Specific  Gravity  of  1032.2 
(water  =  1000)  is  usually  expressed  as  32.2  or,  32.2°  lactometer 
scale. 

Lactometers  indicate  the  specific  gravity  at  a  temperature 
of  60°  F.  and  it  is,  therefore,  necessary  to  either  bring  the  sample 
to  this  temperature  or  to  correct  the  reading.  It  is  much  more 
convenient  to  ascertain  the  temperature  of  the  sample  imme- 
diately before  taking  the  specific  gravity  and  to  correct  this 
result  to  60°  F.  by  means  of  Table  LXVIII,  which  will  be 
found  in  the  appendix. 

It  is  important  that  the  specific  gravity  of  milk  should  not  be 
determined  within  a  short  period  of  milking  as,  during  the  first 
four  hours,  there  is  a  decided  increase  often  amounting  to  1  to 
1.5°  (Recknagel's  phenomenon).  The  gravity  should  also 
never  be  taken  immediately  after  violent  agitation  of  the  sample 
as  the  air  entrapped  by  the  fat  globules  during  such  a  process 
may  lead  to  serious  errors.  If  violent  agitation  is  necessary  for 
any  purpose,  it  is  advisable  to  allow  the  sample  to  remain  quies- 
cent for  two  hours  before  proceeding  with  the  specific  gravity 
determination.  No  attempt  should  be  made  to  take  the  spe- 
cific gravity  of  a  sample  that  has  commenced  to  curdle. 

Total  Solids,  by  Calculation.  As  the  fatty  and  non-fatty 
portions  of  milk  are  comparatively  constant  in  composition, 
it  is  evident  that  the  specific  gravity  of  milk  can  be  calculated 
from  the  percentages  of  these  constituents.  Fat  tends  to  reduce 
the  gravity,  and  non-fatty  solids  to  increase  it.  Hehner  and 
Richmond  found  that  the  following  formula  expressed  with  a 
fair  degree  of  accuracy  the  quantitative  relation  between  these 
constituents: 

F= 0.859  T.  £-0.2186(7. 

Where  F  =  percentage  of  fat,  T.  S.  the  percentage  of  total 
solids  and  G  the  specific  gravity  expressed  as  mentioned  above. 
From  this  formula  T.  £.  =  1.164^+0.2546(7. 

A  simplified  form  of  this  formula  that  has  come  into  general 


MILK  SUGAR  71 

use  is  T.  S.  =  1.2F+Q.25G.  This  is,  with  very  slight  modifi- 
cations, the  basis  of  Babcock's  tables  which  are  official  in  Amer- 
ica. Richmond  now  prefers  the  formula  T.  S.  =  1.2F+0.25(r 
+0.14  and  this  was  used  in  the  preparation  of  the  slide  rule 
which  so  greatly  facilitates  the  calculation  of  the  total  solids 
from  the  fat  and  specific  gravity  determinations.  It  is  ad- 
visable to  remember  that  the  differences  between  the  results 
obtained  by  use  of  the  various  formulae  are  within  the  limits  of 
experimental  error  and  that  a  direct  determination  should  be 
made  when  great  accuracy  is  required. 

Richmond's  and  Babcock's  tables  are  given  in  the  appendix 
on  pages  210-213. 

Solids  Not-fat.  These  are  estimated  by  deducting  the  per- 
centage of  fat  from  that  of  the  total  solids  or  they  may  be  cal- 
culated directly  from  the  gravity  and  the  percentage  of  fat. 

Milk  Sugar.  Milk  Sugar,  or  Lactose,  may  be  estimated  by 
either  the  polarimetric,  volumetric,  or  gravimetric  methods. 
When  a  polarimeter  is  available,  this  method  is  almost  invari- 
ably employed  as  but  little  time  is  required  for  the  examination 
of  several  samples.  In  the  absence  of  this  instrument,  and 
when  only  occasional  determinations  are  required,  the  gravi- 
metric method  should  be  used. 

Polarimetric  Methods.  These  are  based  upon  the  exam- 
ination of  the  milk  serum  in  a  polariscope  after  the  separation 
of  the  fat  and  proteids.  A  solution  of  mercuric  nitrate,  pre- 
pared by  dissolving  mercury  in  twice  its  weight  of  nitric  acid 
(1.42)  and  diluting  with  an  equal  volume  of  water,  is  the  most 
suitable  reagent  for  this  purpose.  As  the  removal  of  proteids 
and  fat  reduce  the  volume  of  the  lactose  containing  solution,  it  is 
necessary  to  correct  the  readings  for  the  percentages  of  these 
constituents,  but  Richmond  and  Boseley  (Dairy  Chemistry) 
point  out  that  these  calculations  can  be  simplified  by  the  use 
of  the  following  method. 

To  100  c.cms.  of  milk  add 

(a)  A  quantity  of  water  in  c.cms.  equal  to  ^  the  lactometer 
reading  or  excess  gravity  over  1000. 


72  CHEMICAL  EXAMINATION 

(6)  A  quantity  of  water  in  c.cms.  equal  to  the  fat  X  1.11. 

(c)  A  quantity  of  water  in  c.cms.  to  reduce  the  scale  readings 
to  percentages  of  milk  sugar. 

(d)  3  c.cms.  of  acid  mercuric  nitrate. 

After  thorough  agitation,  filter  through  dry  papers  and 
polarise  the  filtrate.  The  percentage  of  milk  sugar  can  be  read 
off  directly  in  scale  readings. 

The  values  of  (c)  are: 

(a)  For  polariscopes  reading  angular  degrees. 

With  198.4  mm.  tube  10.0  c.cms. 

With  200  mm.  tube  10.85  c.cms. 

With  500  mm.  tube  10.85  c.cms.  (divide  readings  by  2.5). 

(6)  For  the  Laurent  sugar  scale  (100°  =  21.67  angular  degs.) 
With  200  mm.  tubes  2.33  c.cms.  (divide  readings  by  5) 
With  400  mm.  tubes  2.33  c.cms.  (divide  readings  by  10). 
With  500  mm.  tubes  2.33  c.cms.  (divide  readings  by  12.5) 

(c)  For  the  Ventzke  scale  (100°  =  34.64  angular  degrees). 
With  200  mm.  tube  6.65  c.cms.  (divide  readings  by  3). 
With  400  mm.  tube  6.65  c.cms.  (divide  readings  by  6). 
With  500  mm.  tube  6.65  c.cms.  (divide  readings  by  7.5). 

Gravimetric  Method.  Dilute  25  c.cms.  of  milk  with  400 
c.cms.  of  water  in  a  500  c.cm.  flask,  add  10  c.cms.  of  No.  1, 
Fehling  solution  and  4.4  c.cms.  of  N-NaOH  solution;  make 
up  to  500  c.cms.,  shake,  and  filter  through  a  dry  paper.  The 
filtrate  should  be  acid  and  contain  copper  in  solution.  Place 
25  c.cms.  each  of  Fehling's  solutions  Nos.  1  and  2  in  a  beaker 
and  heat  to  the  boiling  point.  When  boiling  briskly  add  100 
c.cms.  of  the  milk  serum  and  boil  for  six  minutes.  Filter  imme- 
diately through  asbestos,  supported  by  a  platinum  cone  in  a 
hard  glass  filtering  tube,  with  the  aid  of  a  suction  pump,  wash 
thoroughly  with  boiling  water  and  finally  with  alcohol  followed 
by  ether.  After  drying,  connect  the  tube  with  an  apparatus 
for  supplying  a  continuous  current  of  hydrogen  and  gently 
heat  until  the  cuprous  oxide  is  completely  reduced  to  the 


TOTAL  PROTEIDS  73 

metallic  state.  Cool  in  an  atmosphere  of  hydrogen  and  weigh. 
The  weight  of  copper  is  calculated  to  lactose  from  Table 
LXXI  in  the  appendix. 

The  weight  of  lactose  X  20  gives  the  percentage  per  100 
c.cms.  of  sample.  As  an  alternative  method  of  weighing  the 
reduced  oxide,  a  Gooch  crucible  may  be  used  in  which  a  layer 
of  asbestos  about  one-quarter  of  an  inch  in  thickness  has  been 
placed.  Wash  the  asbestos  thoroughly  with  hot  water  and 
then  with  10  c.cms.  of  alcohol  followed  by  10  c.cms.  of  ether. 
Dry  for  thirty  minutes  in  the  steam  oven  and  weigh.  The  pre- 
cipitate of  cuprous  oxide  is  collected  as  above,  washed  with 
water,  treated  with  10  c.cms.  of  alcohol  and  ether,  successively, 
and  dried  for  thirty  minutes  at  100°  C.  The  weight  of  Cu20 
multiplied  by  0.8883  gives  the  weight  of  metallic  copper. 

PKOTEIDS 

Total  Proteids.  5  gms.  of  milk  are  placed  in  a  Kjeldahl 
flask  of  about  150  c.cms.  capacity  and  20  c.cms.  of  pure  cone, 
sulphuric  acid  added.  The  mixture  is  heated  over  a  small  flame 
until  excessive  frothing  has  ceased,  and  after  cooling,  8-10  grms. 
of  acid  potassium  sulphate  and  a  drop  of  mercury  are  added. 
After  placing  a  sealed  funnel  containing  water  in  the  mouth 
of  the  flask  to  prevent  excessive  evaporation,  the  contents  of 
the  flask  are  gradually  heated  and  the  flame  slightly  increased 
as  frothing  ceases.  When  the  liquid  becomes  colourless  the 
flask  is  allowed  to  cool  and  the  contents  washed  with  the  aid  of 
distilled  water  into  a  flask.  This  flask  should  be  provided 
with  a  stopper  having  two  holes,  one  containing  a  trapped  bulb 
tube  connected  with  a  water  condenser,  and  the  other  a  tapped 
funnel  reaching  almost  to  the  bottom  of  the  flask.  After  the 
contents  of  the  Kjeldahl  flask  have  been  transferred,  a  few  pieces 
of-  pumice,  unglazed  porcelain,  or  granulated  zinc,  are  added 
to  prevent  bumping  and  the  distillation  apparatus  connected 
up  with  the  outlet  of  the  condenser  dipping  into  a  beaker  con- 

N 
taining  50  c.cms.  of  -—  acid.     Through  the  funnel  add  100  c.cms. 


74  CHEMICAL  EXAMINATION 

of  30  per  cent  caustic  soda,  followed  by  10  c.cms.  of  a  10  per 
cent  solution  of  potassium  sulphide.  The  flame  is  placed  under 
the  flask,  and  the  distillation  continued  until  about  200  c.cms. 
have  passed  over.  Before  taking  away  the  flame,  the  tap  of 
the  funnel  should  be  opened  to  prevent  creating  a  partial  vac- 
uum and  so  drawing  back  the  distillate  into -the  flask.  The 
end  of  the  condenser  is  washed  with  water,  and  the  washings 

N 
mixed  with  the  distillate  which  is  finally  titrated  with  —  caustic 

alkali  using  sensitive  methyl  orange  or,  preferably,  methyl  red 

N 
as  the  indicator.     Each  c.cm.  of  —  acid  neutralised  =  0.0014 

grm.  nitrogen  or  0.028  per  cent  of  nitrogen  when  5  grms.  of  milk 
are  used.  The  percentage  of  nitrogen  multiplied  by  6.38  gives 
the  percentage  of  total  proteids. 

In  all  determinations  of  nitrogen  by  the  above  method,  it  is 
essential  that  a  blank  determination  should  be  made  on  all  the 
reagents  and  this  amount  deducted  from  all  subsequent  results. 

Caseinogen.  Dilute  10  gms.  of  the  sample  with  about  90 
c.cms.  of  water  at  40°  to  42°  C.  and  add  at  once  1.5  c.cm.  of  a 
10  per  cent  acetic  acid  solution.  Stir  with  a  glass  rod  and  allow 
to  stand  for  about  five  minutes.  Decant  on  to  a  wet  filter, 
wash  several  times  with  cold  water  by  decantation  and  then 
transfer  the  precipitate  completely  to  the  filter.  Wash  once 
or  twice  with  cold  water.  If  the  filtrate  is  not  bright  it  should 
be  refiltered  until  that  condition  is  attained.  The  nitrogen  in 
the  precipitate  is  then  estimated  as  above  by  the  Kjeldahl 
method.  The  percentage  of  nitrogen  multiplied  by  6.38  gives 
the  percentage  of  caseinogen.  This  method  is  only  applicable 
to  fresh  milk. 

Albumin.  The  filtrate  from  the  precipitation  of  caseinogen 
is  first  exactly  neutralised  with  caustic  alkali  and  then  acidified 
by  the  addition  of  0.3  c.cm.  of  a  10  per  cent  solution  of  acetic 
acid.  After  heating  to  boiling  over  a  flame,  the  precipitate  is 
digested  on  the  water  bath  for  fifteen  minutes.  The  liquid  is 
filtered  through  paper,  the  precipitate  washed  and  finally  used 


ALDEHYDE  VALUE  75 

for  a  nitrogen  determination  by  the  Kjeldahl  method.  Nitrogen 
X  6.38  =  Albumin. 

Total  Acidity.  Lactic  Acid.  10  c.cms.  of  milk  are  placed 
in  a  white  porcelain  basin,  a  few  drops  of  phenolphthalein 

N 
solution  added  and  titrated  with  —  alkali  until  a  faint  pink 

colour  is  obtained.  As  the  acidity  of  fresh  milk  is  chiefly  due 
to  phosphates,  the  expression  of  the  acidity  in  terms  of  lactic 
acid  is  somewhat  misleading,  although  this  is  often  done,  1  c.cm. 

N 
of  —  alkali  being  equivalent  to  0.009  grm.  lactic  acid.     It  is 

preferable  to  express  the  acidity  in  degrees,  i.e.,  the  number  of 
cubic  centimeters  of  normal  alkali  required  for  the  neutralisa- 

N 
tion  of  1  litre  of  milk.    The  number  of  cubic  centimeters  of  — 

alkali  required  for  the  neutralisation  of  10  c.cms.  of  milk,  mul- 
tiplied by  10  gives  the  required  result  in  degrees.  It  is  unfor- 
tunate that  in  Germany  the  same  term  is  used  for  a  unit  having 
a  very  different  value.  The  Sohxlet-Henkel  degree  usually 
used  throughout  Germany  is  exactly  2.5  times  greater  than  the 
degree  used  in  England  and  America. 

Aldehyde  Value.  Richmond  and  Miller's  modification 
(Richmond's  Dairy  Chemistry)  of  Steinegger's  method  is  as 
follows:  10  c.cms.  of  milk  are  made  neutral  to  phenolphthalein 

N 
with  —  strontia,  2  c.cms.  of  40  per  cent  formaldehyde  addod, 

and  again  titrated  to  the  same  degree  of  neutrality.  The 
amount  of  the  second  addition  of  alkali  less  the  amount  re- 
quired for  the  neutralisation  of  the  formaldehyde  added  (pre- 
viously determined),  multiplied  by  10  gives  the  aldehyde  value. 
This  method  is  dependent  upon  the  fact  that  the  proteid 
radicle  is  quantitatively  converted  to  an  acid  by  the  aldehyde. 
Richmond  states  that  the  strontia  aldehyde  figure  is  1.1  times 

N 
greater  than  that  given  with  —  soda  and  that  the  former  value 

multiplied  by  0.170  will  give  a  close  approximation  to  the  total 


76  CHEMICAL  EXAMINATION 

proteids.  It  is  also  pointed  out  that  as  caseinogen  and  albumin 
do  not  give  the  same  aldehyde  value,  the  factor  is  only  applica- 
ble when  the  ratio  of  caseinogen  to  albumin  is  normal. 

Mineral  Constituents.  The  estimation  of  the  mineral 
constituents  in  milk  is  but  infrequently  required  in  connection 
with  public  health  work  but  on  these  occasions,  the  following 
method,  due  to  Droop  Richmond,  will  be  found  advantageous 
as  it  secures  fairly  accurate  results  with  a  minimum  expenditure 
of  time  and  labour. 

Fifty  grams  of  milk  are  evaporated  and  charred  to  a  black 
ash :  the  mass  is  extracted  with  hot  water  and  filtered,  the  insol- 
uble portion,  together  with  the  paper  (after  washing)  being 
ignited  until  white;  this  gives  the  insoluble  ash.  Evaporation 
of  the  filtrate  and  cautious  heating  gives  the  weight  of  the  sol- 
uble ash. 

The  soluble  ash,  after  solution  in  water,  is  made  up  to  a 
known  volume  and  aliquot  portions  used  for  the  determination 

N 
of  the  alkalinity  by  titration  with  —  acid  with  methyl  orange 

N 
as  indicator,  and  chlorine  by  titration  with  —  silver  nitrate, 

N 
using  potassium  chromate  as  indicator.     1  c.cm.  of  —  acid 

=  0.0031  grm.  Na20  and  1  c.cm.  ^  AgN03  =  0.00355  grm.  Cl. 

The  insoluble  ash  is  dissolved  in  a  slight  excess  of  dilute 
hydrochloric  acid,  and  the  solution  (nearly  neutralised  if  nec- 
essary) heated  to  boiling;  a  cold  saturated  solution  of  ammo- 
nium oxalate  is  dropped  in  slowly  until  further  addition  pro- 
duces no  further  precipitate.  After  standing  at  least  two  hours, 
the  precipitate  is  filtered  off,  washed,  and  ignited  at  a  low  tem- 
perature to  convert  the  oxalate  into  carbonate;  it  is  advisable 
to  moisten  the  ignited  precipitate  with  ammonium  carbonate 
solution  and  reignite  at  a  very  low  temperature.  The  precipi- 
tate, after  weighing,  is  dissolved  in  dilute  hydrochloric  acid, 
keeping  the  bulk  small,  ammonia  is  added  to  alkaline  reaction, 


MINERAL  CONSTITUENTS  77 

and  the  small  precipitate  of  calcium  phosphate  collected,  ignited, 
and  weighed.  Its  weight  is  subtracted  from  the  previous 
weight,  and  the  difference  gives  the  weight  of  calcium  carbonate, 
which,  multiplied  by  0.4,  gives  the  calcium,  or  by  0.56,  the  lime 
(CaO)  contained  in  it;  the  weight  of  calcium  phosphate  mul- 
tiplied by  0.3871  gives  the  calcium  (Ca),  or  by  0.5419,  the  lime 
(CaO)  contained  in  it.  The  total  calcium  or  lime  is  the  sum  of 
the  two. 

The  filtrate  is  made  strongly  ammoniacal  by  the  addition  of 
strong  ammonia  (0.880)  and  allowed  to  stand  twenty-four  hours. 
The  precipitated  magnesium  ammonium  phosphate  is  filtered 
off,  washed  with  dilute  ammonia,  ignited,  and  the  magnesium 
pyrophosphate  (Mg2?207)  weighed.  Its  weight  multiplied 
by  0.2162  will  give  the  magnesium  (Mg),  or  by  0.3604,  the 
magnesia  (MgO)  contained  in  it. 

To  the  filtrate  from  this,  magnesia  mixture  is  added,  and 
the  precipitate,  after  standing  twenty-four  hours,  is  treated 
as  above.  From  the  total  weight  of  the  two  quantities  of  mag- 
nesium pyrophosphate,  the  phosphoric  anhydride  is  calculated 
by  multiplying  by  0.6396;  to  this  is  added  the  phosphoric  anhy- 
dride in  the  calcium  phosphate,  calculated  by  multiplying 
the  weight  by  0.4581.  This  method  takes  no  account  of  the 
traces  of  iron  present,  which  are  precipitated  with  the  calcium 
phosphate  and  the  magnesium-ammonium  phosphate.  If 
desired,  this  may  be  estimated  by  dissolving  the  precipitate  of 
calcium  phosphate  and  the  first  magnesium-ammonium  phos- 
phate precipitate  in  dilute  hydrochloric  acid,  and  determining 
the  iron  colorimetrically  as  thiocyanate. 

To  estimate  alkalies,  another  portion  of  milk  is  ignited  as 
before,  and  the  total  ash  dissolved  in  dilute  hydrochloric  acid 
and  boiled;  a  few  drops  of  barium  chloride  solution,  containing 
not  more  than  0.1  grm.  of  barium  to  100  grms.  of  milk  are 
added,  and  the  boiling  continued  for  some  minutes.  After  some 
hours,  the  precipitate  of  barium  sulphate  is  filtered  off,  washed, 
ignited,  and  weighed;  its  weight  multiplied  by  0.3433,  will 
give  the  sulphuric  anhydride  (SOs)  in  the  milk.  If  an  excess 


78  CHEMICAL  EXAMINATION 

of  barium  chloride  has  been  added,  a  little  phosphoric  acid,  or 
ammonium  phosphate,  may  now  be  added  to  the  filtrate, 
although  it  is  not  necessary  if  the  quantity  of  barium  chloride 
given  above  has  been  employed.  A  quantity  of  ferric  chloride 
solution,  sufficient  to  colour  the  solution  brown,  is  added  and 
the  filtrate  made  alkaline  with  ammonia.  After  boiling,  the 
precipitate  is  filtered  off  and  well  washed:  the  filtrate  is  evap- 
orated and  cautiously  ignited:  this  weight  represents  the  alka- 
line chlorides.  When  the  residue  is  dissolved  in  hot  water,  the 
solution  should  be  perfectly  clear;  if  this  be  not  so,  a  little 
ammonium  carbonate  solution  is  added,  the  liquid  evaporated 
to  dryness  and  the  residue  cautiously  ignited;  the  residue  is 
again  taken  up  with  water,  the  solution  filtered  and  evaporated, 
and  the  residue  cautiously  ignited  and  weighed.  This  puri- 
fication of  the  mixed  alkaline  chlorides  is  often  found  necessary 
and  it  is  essential,  in  order  that  accurate  results  may  be  obtained, 
that  the  process  should  be  carried  out  with  great  care,  always 
bearing  in  mind  that  alkaline  chlorides  are  volatilised  at  com- 
paratively low  temperatures. 

The  chlorine  in  the  mixed  chlorides  may  be  estimated  by 

N 
titration  with  — -  silver  nitrate,  using  potassium  chromate  as 

N 
indicator.    Each  cubic  centimeter  of  —  AgNOs  is  equivalent 

to  0.00355  grm.  chlorine.  The  potassium  and  sodium  are  cal- 
culated from  the  formulae. 

The  weight  of  sodium      =  2.997C  -  1.4254W, 
The  weight  of  potassium  =  2.4254W-3.987C. 

in  which  W  =  the  weight  of  the  mixed  alkaline  chlorides, 

and          C=the  weight  of  chlorine  therein. 

Examination  of  Milk  Serum.  As  the  fat  and  proteids  are 
the  most  variable  constituents  of  milk,  an  examination  of  the 
milk  serum  often  affords  valuable  assistance  in  determining 


EXAMINATION  OF  MILK  SERUM 


79 


whether  a  sample  is  adulterated  by  the  addition  of  water,  or  is 
merely  abnormal  in  composition.  The  principal  constituents 
of  the  serum  are  milk  sugar  and  mineral  matter,  and  a  deter- 
mination of  these  on  the  milk  direct  affords  the  same  evidence  as 
an  indirect  examination  of  the  serum,  but  as  the  latter  can  be 


TABLE  XXXV 
RELATION  OF  REFRACTIVE  INDEX  TO  SPECIFIC  GRAVITY 

(LYTHGOE) 


Scale  Reading  Immersion 
Refractometer.     20°  C. 

nD  20°  C. 

Specific  Gravity. 
15° 
15°' 

28.0 

1.33820 

1.0149 

29.0 

1  .33861 

1.0160 

30.0 

1.33896 

1.0170 

31.0 

1.33934 

1.0180 

32.0 

1.33972 

1.0190 

33.0 

1.34010 

1.0200 

34.0 

1.34048 

1.0211 

35.0 

1.34086 

1.0221 

36.0 

.34124 

1.0231 

37.0 

.34162 

1.0242 

38.0 

.34199 

.0252 

39.0 

.34237 

.0262 

40.0 

.34275 

.0273 

41.0 

.34313 

.0283 

42.0 

.34350 

.0293 

43.0 

.34388 

.0303 

44.0 

.34426 

.0313 

45.0 

1.34463 

.0323 

performed  more  expeditiously,  it  is  often  included  in  the  rou- 
tine examination  of  milk.  The  serum  is  prepared  by  adding 
2  c.cms.  of  25  per  cent  acetic  acid  (Sp.  Gr.  1.035)  to  100  c.cms. 
of  sample  at  a  temperature  of  20°  C.,  covering  with  a  watch- 
glass  and  heating  to  70°  C.  for  twenty  minutes.  After  cooling 
in  ice  water  for  ten  minutes,  the  curd  is  separated  by  filtration 


80  CHEMICAL  EXAMINATION 

through  paper  and  35  c.cms.  of  the  filtrate,  which  should  be 
bright,  are  transferred  to  one  of  the  beakers  which  accompany 
the  Zeiss  immersion  refractometer.  The  refraction  is  then 
determined  at  exactly  20.0°  C.  A  reading  between  39.0  and 
40.0  is  suspicious  whilst  one  less  than  39.0  indicates  the  addition 
of  water. 

Lythgoe1  after  determining  the  value  of  K  in  the  Lorenz  and 
Lorentz  formula 

n2-l  d_K 

ft2+2*l~ 

which  expresses  the  relation  between  the  refractive  index  (ft) 
and  the  specific  gravity  (d),  has  calculated  the  values  of  d  for 
the  various  scale  readings  of  the  immersion  refractometer,  and 
in  the  absence  of  this  instrument,  the  specific  gravity  deter- 
mination will  achieve  the  same  object  after  reference  to  Lyth- 
goe's  table.  (Table  XXXV,  p.  79.) 

DETECTION  AND  ESTIMATION  OF  PRESERVATIVES 

The  addition  of  preservatives  to  milk  is  usually  absolutely 
prohibited  because  it  has  been  found  perfectly  feasible  to  market 
this  product  in  a  sound  condition  without  then-  use.  No  legit- 
imate excuse,  therefore,  for  the  addition  of  any  substance  which 
retards  or  inhibits  bacterial  development.  Although  the  exig- 
encies of  certain  branches  of  trade  in  milk  products  have,  in 
some  cases,  led  to  the  adoption  of  regulations  which  permit 
the  addition  of  certain  specified  preservatives  in  quantities 
not  exceeding  a  specified  limit,  this  practice  should  not  be 
encouraged,  for,  until  it  can  be  proved  beyond  reasonable 
doubt  that  such  preservatives  are  non-toxic,  the  public  should 
be  safeguarded  against  these  substances:  public  health  should 
be  paramount  to  commercial  interests  and  not  sacrificed  to 
them.  Unfortunately  many  statutes  regarding  the  sophis- 
tication of  foodstuffs  are  even  yet  so  framed  as  to  place  the  onus 
of  proof  as  to  damage  to  health  upon  the  prosecutor  and  so  give 
the  defendant  the  benefit  of  all  doubts  that  may  exist,  but  it  is 
pleasing  to  note  that  these  are  decreasing  and  that  the  present 


FORMALDEHYDE  81 

tendency  is  to  prohibit  the  entire  use  of  particular  preservatives 
and  to  restrict  them  generally. 

The  preservatives  in  most  general  use  are  boric  acid,  borax, 
or  mixtures  of  these  two,  and  formaldehyde.  For  milk  the  last- 
mentioned  is  the  favourite  owing  to  its  potency  and  general  con- 
venience. The  presence  of  boric  acid  or  borax  is  allowed  in 
cream  in  England  when  declared  on  the  label  attached  to  the 
container  and  in  quantities  not  exceeding  0.25  per  cent  when 
calculated  as  boric  acid.  Harden  has  shown  that  the  addition 
of  an  alkali  (7  grms.  of  Na20  per  100  grms.  of  boric  acid)  in- 
creases the  efficiency  of  boric  acid  as  a  preservative,  and  it  is 
now  customary  to  employ  such  a  mixture  for  the  preservation  of 
cream.  Such  mixtures  also  contain  cane  sugar  or  traces  of 
saccharin,  the  object  of  which  is  to  mask  incipient  sourness. 

Formaldehyde.  Formaldehyde  may  be  detected  by  any 
of  the  following  tests,  but  on  account  of  its  reliability  and  del- 
icacy, the  author  recommends  the  Shrewsbury  and  Knapp 
process. 

Hehner  Method.  About  10  c.cms.  of  sample  are  placed 
in  a  test  tube  and  concentrated  commercial  sulphuric  poured 
carefully  down  the  side  so  as  to  form  a  layer  beneath  the  milk. 
In  the  presence  of  formaldehyde,  a  violet  ring  is  formed  at  the 
junction  of  the  two  liquids.  Richmond  and  Boseley  modified 
the  test  by  adding  an  equal  volume  of  water  to  the  milk  and 
using  acid  of  90  to  94  per  cent  strength.  One  part  in  200,000 
produces  a  violet  colouration  which  is  permanent  for  several 
days.  In  the  absence  of  formaldehyde,  a  greenish  ring  is  pro- 
duced and  a  brick-red  colouration  in  the  acid  layer. 

Leonard2  points  out  that  the  presence  of  a  mild  oxidising 
agent  is  essential  for  the  success  of  this  test  and  that  such  an 
agent,  preferably  a  trace  of  ferric  chloride,  must  be  added  if 
pure  acid  is  used.  Droop  Richmond  3  points  out  that  the  test 
is  dependent  upon  the  reaction  of  formaldehyde  with  the 
tryptophane  of  the  caseinogen  and  that  other  aldehydes,  e.g., 
vanillin,  give  similar  reactions. 

Hydrochloric  Acid    Test.     10  c.cms.  of  commercial  hydro- 


82  CHEMICAL  EXAMINATION 

chloric  acid,  containing  0.2  grm.  of  ferric  chloride  per  litre, 
are  added  to  5  c.cms.  of  milk  in  a  porcelain  basin  and  the  mix- 
ture heated  to  boiling  with  constant  stirring.  The  presence  of 
formaldehyde  is  indicated  by  a  violet  colouration. 

Shrewsbury  and  Knapp  Test.4  The  reagent  for  this 
test  consists  of  a  freshly  prepared  mixture  of  pure  concentrated 
hydrochloric  acid  with  0.1  per  cent  of  pure  nitric  acid.  5  c.cms. 
of  the  sample  are  placed  in  a  test  tube  and  vigourously  shaken 
with  10  c.cms.  of  the  reagent,  the  mixture  is  heated  in  a  water 
bath  to  50°  C.  for  ten  minutes  and  finally  rapidly  cooled  to 
about  15°  C.  A  violet  colouration  denotes  the  presence  of 
formaldehyde,  and  a  rose  pink  colouration,  its  absence.  The 
depth  of  the  colouration,  between  0.2  and  6  parts  per  million, 
is  approximately  proportional  to  the  amount  of  formaldehyde 
present,  so  that  this  method  may  also  be  used  for  the  estimation 
of  the  preservative.  When  the  amount  exceeds  six  parts  per 
million,  the  milk  should  be  suitably  diluted. 

Estimation  of  Formaldehyde.  In  addition  to  the  method 
previously  mentioned,  various  others  have  been  devised  for  the 
estimation  of  formaldehyde,  but  not  one  as  yet  can  be  relied 
upon  to  give  accurate  results.  Most  of  these  are  based  upon  the 
volatilisation  of  the  aldehyde  by  distillation  of  an  acid  solution, 
and  subsequent  volumetric  estimation.  Probably  the  most 
useful  is  the  following.  To  100  c.cms.  of  sample  contained  in  a 
500  c.cm.  Kjeldahl  flask  add  1  c.cm.  of  1  :  3  sulphuric  acid  and 
distil  over  20  c.cms.  (care  is  necessary  if  frothing  is  to  be 
avoided).  The  formaldehyde  in  the  distillate,  amounting  to 
approximately  one-third  of  the  total,  is  estimated  iodometrically. 

N 
25  c.cms.  of  —  iodine  solution  are  added  to  the  distillate  and 

normal  caustic  soda  is  added,  drop  by  drop,  until  the  liquid 
becomes  a  clear  yellow.  After  standing  for  fifteen  minutes, 
dilute  sulphuric  acid  is  added  in  excess  to  liberate  the  uncom- 

N 
bined  iodine.     The  solution  is  then  titrated  with  —  sodium 

thiosulphate,  using  a  starch  solution  as  the  indicator  in  the  end 


BORIC  ACID  AND  BORATES  83 

N  . 
reaction.    Each  cubic  centimetre  of  —  iodine  solution  absorbed 

equals  0.0015  grm.  of  formaldehyde. 

Monier- Williams,  in  a  report  to  the  Local  Government 
Board,  states  that  a  preservative  is  on  the  market  which  con- 
tains a  nitrite  in  addition  to  formaldehyde:  the  nitrite  masks 
the  usual  reactions  but  its  effect  may  be  destroyed  by  the 
addition  of  a  little  urea. 

Boric  Acid  and  Borates.  These  may  be  detected  by  adding 
a  few  cubic  centimetres  of  normal  alkali  to  not  less  than  10 
cubic  centimetres  of  milk  and  evaporating  to  dryness  over  a 
small  flame.  The  flame  is  increased  until  a  black  ash  results: 
this  is  acidified  with  a  few  drops  of  hydrochloric  acid.  After 
lixiviation  with  a  few  cubic  centimetres  of  hot  water,  the  ash  is 
removed  by  filtration  through  paper.  A  turmeric  paper  is 
placed  in  the  filtrate  in  such  a  manner  that  only  a  portion  of  it 
can  be  wetted,  and  the  liquid  evaporated  to  dryness.  A  red- 
dish-brown colouration  of  the  wetted  portion,  due  to  the  for- 
mation of  rosocyanin,  indicates  the  presence  of  boron  com- 
pounds. A  drop  of  caustic  soda  changes  the  colouration  to 
various  shades  of  green  and  purple  which  can  be  restored  to 
the  original  colour  by  the  addition  of  hydrochloric  acid. 

A  useful  routine  method  for  the  detection  of  boron  com- 
pounds consists  in  heating  about  10  c.cms.  of  milk  in  a  porce- 
lain dish  with  a  few  cubic  centimetres  of  methyl  alcohol  and  a 
few  drops  of  tincture  of  turmeric.  The  heating  is  conveni- 
ently carried  out  in  a  water  bath  and  the  presence  of  boron 
compounds  is  indicated  by  the  formation  of  a  reddish  ring 
round  the  basin. 

The  estimation  of  boron  compounds  is  most  conveniently 
carried  out  by  Thomson's  method.5  One  or  two  cubic  centi- 
metres of  N-NaOH  are  added  to  100  c.cms.  of  milk  and  the 
whole  evaporated  to  dryness  in  a  platinum  dish.  The  residue 
is  ignited  to  a  black  ash,  heated  with  20  c.cms.  of  water  and 
concentrated  hydrochloric  acid  added,  drop  by  drop,  until 
frothing  ceases.  The  solution  containing  the  carbonaceous 


84  CHEMICAL  EXAMINATION 

matter  is  washed  with  a  few  cubic  centimetres  of  water  into  a 
100  c.cm.  flask  and  0.5  grm.  dry  calcium  chloride  added.  After 
the  addition  of  a  few  drops  of  phenolphthalein,  a  10  per  cent 
solution  of  caustic  soda  is  added  until  a  faint  pink  colour  per- 
sists and  finally  25  c.cms.  of  lime  water.  The  object  of  this  is 
to  precipitate  the  phosphates  as  calcium  phosphate.  Make  up 
the  volume  to  100  c.cms.,  mix  thoroughly  and  filter  through  a 
dry  paper.  To  50  c.cms.  of  the  filtrate  add  N.  sulphuric  acid 
until  just  colourless,  then  add  a  few  drops  methyl  orange  and 
continue  the  titration  until  the  yellow  colour  changes  to  pink. 

N 

—  soda  is  now  added  until  the  reaction  is  just  alkaline  and  the 

liquid  boiled  to  expel  the  carbonic  acid  liberated.  The  solu- 
tion is  cooled,  a  few  drops  of  phenolphthalein  solution  and 
sufficient  neutral  glycerine  to  amount  to  40  per  cent  of  the  total 

N 
volume  is  added.    The  solution  is  finally  titrated  with  —  soda 

until  a  permanent  pink  colouration  is  produced.    Each  cubic 

N 
centimetre  of  —r  soda  equals  0.0062  grm.  of  boric  acid. 

Benzole  Acid.  The  proteids  are  precipitated  by  the  addi- 
tion of  5  c.cms.  of  dilute  hydrochloric  acid  and  shaking:  then 
shake  with  several  portions  of  ether,  taking  care  to  avoid  the 
formation  of  an  emulsion.  If  this  should  occur,  resort  must  be 
made  to  a  centrifuge  in  order  to  separate  it.  The  ethereal 
extract  containing  the  benzoic  acid  and  fat  is  shaken  with  water, 
rendered  alkaline  by  the  addition  of  ammonia,  and  the  aqueous 
extract  evaporated  nearly  to  dryness.  After  all  the  ammonia 
has  disappeared,  a  few  drops  of  ferric  chloride  are  added  and 
the  presence  of  benzoic  acid  is  indicated  by  the  formation  of  a 
flesh-coloured  precipitate.  If  any  ammonia  is  left  in  the  solu- 
tion, a  reddish-brown  precipitate  of  ferric  hydrate  is  obtained, 
so  that  it  is  essential  that  all  traces  of  this  disturbing  sub- 
stance are  removed  before  applying  the  final  test. 

Salicylic  Acid.  This  is  detected  in  exactly  the  same  manner 
as  is  described  above  for  the  detection  of  benzoic  acid.  On  addi 


DETECTION  OF  ADDED  COLORING  MATTER  85 

tion  of  ferric  chloride,  a  solution  of  salicylic  acid  produces  a 
characteristic  violet  colour,  the  intensity  of  which  is  somewhat 
proportional  to  the  amount  of  salicylic  acid  present. 

Hydrogen  Peroxide.  As  hydrogen  peroxide  decomposes 
into  free  oxygen  and  water  soon  after  its  addition  to  milk,  it  is 
impossible  to  detect  this  substance  by  means  of  the  usual 
reagents.  The  oxygen  liberated,  however,  considerably  mod- 
ifies the  enzymes  present,  and  it  is  upon  this  fact  that  several 
inferential  tests  for  detecting  hydrogen  peroxide  are  based. 
The  immediate  reductase  reaction  (see  p.  89)  is  destroyed  by 
hydrogen  peroxide,  and  the  catalase  (see  p.  91)  destroyed  in 
proportion  to  the  amount  added. 

Before  the  hydrogen  peroxide  has  decomposed  it  may  be 
detected  by  the  peroxidase  reaction  (see  p.  91). 

Hypochlorites.  Although  hypochlorites  have  been  sug- 
gested as  milk  preservatives  they  have  not  been  extensively 
used  as  the  amount  required  to  produce  any  appreciable  effect 
also  adversely  affects  the  taste  and  odour.  Milk  containing 
hypochlorites  does  not  give  the  usual  starch-iodide  reaction 
even  with  as  large  a  quantity  as  50  parts  of  available  chlorine 
per  100,000. 

Detection  of  Added  Colouring  Matter.  The  following  are 
the  provisionally  official  methods  of  the  American  Association 
of  Official  Agricultural  Chemists. 

Warm  about  150  c.cms.  of  milk  in  a  basin  over  a  flame  and 
add  about  5  c.cms.  of  acetic  acid,  after  which  slowly  continue 
the  heating  almost  to  the  boiling  point  whilst  stirring.  Gather 
the  curd,  when  possible,  into  one  mass  by  means  of  the  stirring 
rod,  and  pour  off  the  whey.  If  the  curd  breaks  up  into  small 
flecks,  separate  from  the  whey  by  straining  through  a  sieve  or 
muslin.  Press  the  curd  free  from  adhering  liquid,  transfer  to  a 
small  flask,  and  macerate  for  several  hours  (preferably  over- 
night) in  about  50  c.cms.  of  ether,  the  flask  being  tightly  corked 
and  shaken  at  intervals.  The  ether  is  finally  decanted  from  the 
curd  and  is  examined  for  annatto,  the  curd  being  reserved  for 
the  detection  of  aniline  orange  and  caramel. 


86  CHEMICAL  EXAMINATION 

Annatto.  After  evaporation  of  the  ether,  the  fatty  residue 
is  made  alkaline  with  caustic  soda  and,  whilst  still  warm, 
poured  upon  a  very  small  wet  filter  paper.  After  the  solution 
has  passed  through,  wash  the  fat  from  the  paper  with  a  stream 
of  water  and  dry  the  paper.  If,  after  drying,  the  paper  is 
coloured  orange,  the  presence  of  annatto  is  indicated.  This 
may  be  confirmed  by  adding  a  drop  of  stannous  chloride  solu- 
tion, which,  in  the  presence  of  annatto,  produces  a  character- 
istic pink  on  the  orange-coloured  paper. 

Aniline  Orange.  The  curd  of  an  uncoloured  milk  is  per- 
fectly white  after  complete  extraction  with  ether,  as  is  also 
that  of  a  milk  coloured  with  annatto.  If  the  extracted  curd  is 
distinctly  dyed  an  orange  or  yellowish  colour,  the  presence  of 
aniline  orange  is  indicated.  To  confirm  this,  treat  a  lump  of 
the  fat-free  curd  with  a  little  strong  hydrochloric  acid.  If  the 
curd  turns  pink,  the  presence  of  aniline  orange  is  assured. 

Aniline-  orange  may  also  be  detected  by  Lythgoe's  method 
which  consists  of  the  addition  of  10  c.cms.  of  concentrated 
hydrochloric  acid  to  an  equal  volume  of  milk  in  a  porcelain 
dish  and  imparting  a  rotary  motion  to  the  contents.  If  any 
appreciable  amount  of  aniline  orange  is  present,  a  pink  colour 
is  at  once  imparted  to  the  curd  particles  as  they  separate. 

Caramel.  If  the  fat-free  curd  is  coloured  a  dull  brown, 
caramel  is  suspected.  Shake  a  lump  of  the  curd  with  concen- 
trated hydrochloric  acid  in  a  test  tube  and  heat  gently.  In 
the  presence  of  caramel  the  acid  solution  will  gradually  turn  a 
deep  blue,  as  will  also  the  white  fat-free  curd  of  an  uncoloured 
milk,  while  the  curd  itself  does  not  change  colour.  It  is  only 
when  this  blue  colouration  of  the  acid  occurs  in  conjunction  with 
a  brown-coloured  curd,  which  itself  does  not  change  colour, 
that  caramel  can  be  suspected,  as  distinguished  from  the  pink 
colouration  produced  by  aniline  orange  under  similar  circum- 
stances. 


CREAM  87 

ANALYSIS  OF  MILK  PRODUCTS 

Cream.  The  normal  constituents  can  be  determined  by 
employing  the  usual  methods  of  milk  analysis  after  suitable 
detection  with  water  (vide  p.  66).  The  amount  of  cream 
used  for  dilution,  however,  should  be  weighed  and  not  measured 
volumetrically.  The  total  solids  should  be  determined  by 
evaporation,  and  Richmond  recommends  the  addition  of  an 
equal  volume  of  alcohol  to  accelerate  drying.  Richmond  also 
finds  that  the  total  solids  and  fat  bear  the  relation  expressed  by 
the  formula: 

Fat  =  1.102  Total  Solids -10.2 

Thickening  agents  are  sometimes  added  to  cream  for  the 
purpose  of  increasing  the  viscosity  and  thus  produce  the  appear- 
ance of  a  cream  of  high  fat  content.  The  usual  agents  employed 
are  gelatine,  starch,  and  saccharate  of  lime  (viscogen). 

Small  quantities  of  gelatine  may  be  detected  by  Stokes' 
method.6  Mercury  is  dissolved  in  twice  its  weight  of  con- 
centrated nitric  acid  (1.42)  and  the  solution  diluted  with 
twenty-five  times  its  volume  of  water.  To  10  c.cms.  of  cream 
add  an  equal  bulk  of  mercuric  nitrate  solution  and  about  20 
c.cms.  of  cold  water.  Shake  vigourously  and  filter  after  stand- 
ing for  a  few  minutes.  Inability  to  obtain  a  clear  filtrate  indi- 
cates the  presence  of  gelatine  and  this  may  be  confirmed  by 
adding  an  equal  volume  of  a  saturated  solution  of  picric  acid. 
A  yellow  precipitate  is  produced  by  gelatine  in  a  cold  solution. 

Starch  is  detected  by  the  formation  of  a  blue  colouration  on 
addition  of  a  solution  of  iodine  in  potassium  iodide. 

Saccharate  of  lime  may  be  detected  by  the  estimation  of 
either  the  lime  in  the  ash  or  by  the  lactose  determination.  The 
lime  in  normal  samples  averages  about  22.4  per  cent  of  the  ash 
and  any  perceptible  increase  over  this  amount  is  suspicious. 
Similarly  an  abnormally  high  polarimeter  reading,  equivalent, 
when  calculated  as  lactose,  to  more  than  52.5  per  cent  of  the 
solids  not  fat,  should  also  be  regarded  with  suspicion. 


88  CHEMICAL  EXAMINATION 

Skim  Milk.  The  usual  methods  of  milk  analysis  may  be 
applied. 

Condensed  Milk.  About  30  grins,  of  milk  are  weighed  out 
and,  after  boiling  with  50  c.cms.  of  water,  the  solution  is  cooled 
and  made  up  to  100  c.cms.  The  methods  of  analysis  described 
above  under  milk  may  then  be  applied,  but  longer  extraction 
should  be  given  if  the  Adams  process  is  used  for  the  estimation 
of  the  fat. 

In  sweetened  samples  the  cane  sugar  is  determined  by  sub- 
tracting the  sum  of  the  fat,  lactose,  proteids,  and  ash,  from  the 
total  solids. 

ENZYMES 

Although  the  presence  of  enzymes  in  milk  has  been  an 
established  fact  for  many  years,  it  is  only  comparatively  recently 
that  the  origin  of  these  ferments  has  been  seriously  considered. 
The  nature  and  characteristics  of  these  bodies  suggests  that 
they  are  derived  from  the  blood  and  the  results  of  various 
experimenters  show  that  they  are  largely  associated  with  the 
cells  invariably  found  in  milk  samples.  Whilst  the  greater 
portion  of  the  enzyme  activity  of  milk  is  anchored  to  the  cells 
and  may,  consequently,  be  removed  by  nitration,  there  is  also 
present  a  smaller  quantity  of  extra  cellular  activity.  This  is 
not  surprising  when  the  rapid  metabolic  changes  taking  place 
during  the  secretion  of  milk  are  considered.  Certain  enzymes, 
such  as  Schardinger's  reductase,  occur  in  amounts  which  vary 
directly  with  the  fat  content  and,  unless,  this  enzyme  is  almost 
entirely  extra  cellular,  the  cells  should  also  vary  somewhat  with 
the  fat  content.  Although  various  hypotheses  have  been  ad- 
vanced as  to  the  effect  of  enzymes  in  milk,  the  author  believes 
that  too  much  importance  has  been  attached  to  the  qualitative 
and  too  little  to  the  quantitative  tests  for  these  substances. 
The  amylase  content  of  normal  milk  is  equivalent  to  about  0.4 
grm.  of  starch  per  100  c.cms.  of  milk  per  hour.  The  catalase 
in  100  c.cms.  liberates  from  hydrogen  peroxide  10  c.cms.  or 
0.014  grm.  of  oxygen  in  two  hours,  whilst  Babcock  and  Russell's 


ENZYMES  89 

figures  show  the  galactase  activity  to  be  capable  of  digesting 
approximately  1  per  cent  of  proteids  in  milk  in  twenty-four 
hours.  Compared  with  the  activity  of  the  normal  secretions 
of  the  alimentary  tract,  these  quantities  are  so  small  as  to  pos- 
sess but  little,  if  any,  physiological  significance.  Pathological 
conditions  such  as  mastitis,  which  involve  inflammatory  pro- 
cesses of  the  udder,  increase  the  cell  content  and,  consequently, 
also  the  enzyme  activity  of  milk,  whilst  heating  of  the  milk  to 
temperatures  of  75°  C.  and  over,  weaken  and  finally  destroy 
the  enzymes.  As  an  aid  to  the  diagnosis  of  such  conditions  and 
for  the  control  of  pasteurisation,  the  determination  of  the  fer- 
ment activity  may  be  found  desirable  and  for  this  purpose  the 
following  methods  have  been  proved  to  be  satisfactory.  The 
determinations  that  can  be  most  conveniently  carried  out  in 
routine  work  and  which  do  not  require  special  apparatus  are 
the  reductase  and  peroxide  tests:  the  catalase  and  amylase 
follow  next  in  order  of  facility  whilst  the  others  are  of  more 
scientific  interest  than  practical  utility. 

Reductase.  To  10  c.cms.  of  milk,  add  1  c.cm.  of  Schar- 
dinger's  reagent  (190  parts  water  and  5  parts  each  of  formalin 
and  a  saturated  alcoholic  solution  of  methylene  blue)  and  heat 
to  43°-45°  C.:  the  time  required  for  decolourisation  is  noted. 
The  reoxidation  of  the  surface  layers  by  the  air  may  be  entirely 
prevented  by  adding  a  small  quantity  of  paraffin,  but  the 
cream  layer  usually  gives  the  necessary  protection. 

Any  desired  temperature,  not  exceeding  60°  C.,  may  be 
used  for  carrying  out  this  test,  but  whatever  temperature  is 
chosen  must  be  adhered  to  in  order  that  the  results  may  be 
strictly  comparative.  In  most  laboratories,  a  temperature  of 
43°-45  C.  will  be  found  convenient  as  the  water  bath  employed 
for  liquid  agar  media  is  usually  maintained  at  this  temperature. 

This  ferment  is  not  present  in  every  sample  of  milk  from 
individual  cows,  being  frequently  absent  from  animals  whose 
offspring  are  still  suckling  and  in  animals  whose  lactation  period 
is  just  commencing  (Schern)  but  the  author  has  invariably 
found  it  to  be  present  in  mixed  market  samples.  Homer  and 


90 


CHEMICAL  EXAMINATION 


Sames  have  found  that  it  does  not  decolourise,  or  only  com- 
pletely so,  in  the  fore  milk  and  that  the  time  required  for 
decolourisation  becomes  less  as  the  milking  proceeds.  This 
corresponds  to  the  relative  frequency  of  the  fat  content  and 
on  this  connection  the  following  figures  calculated  from  some  of 
the  author's  results  are  of  interest: 


TABLE  XXXVI 
RELATION  OF  BUTTER  FAT  TO  REDUCTASE  CONTENT 


Butter  Fat  Content. 

Average  Time  for  Reduction. 

Minutes. 

Less  than  3.  4 

15 

3.4to3.6 

17 

3.7to3.9 

16 

4.0to4.2 

14 

4.3to4.5 

13 

4.6to4.8 

10 

More  than  4.9 

7 

The  following  results  of  the  author  show  that  there  is  no 
relation  between  the  bacterial  content  of  milk  and  the  reductase 
test  or  hastened  reductase  test  as  it  is  sometimes  known  as 
(cf.  p.  24): 

TABLE  XXXVII 
RELATION  OF  BACTERIAL  COUNT  TO  REDUCTASE  CONTENT 


Bacterial  Count  per  C.cm. 
Agar  48  Hrs.  at  37°  C. 

Average  Terms  of  Reduction. 
Minutes. 

Less  than         10,000 

13 

10,001  to    50,000 

16 

50,001  to  100,000 

14 

100,001  to  200,000 

17 

200,001  to  300,000 

17 

300,001  to  400,000 

19 

AMYLASE  91 

Peroxidases.  The  detection  of  this  ferment  may  be  carried 
out  by  any  of  the  following  methods,  all  of  which  are  reliable. 

Rothenfusser's  Method.  Two  solutions  are  required:  (1) 
a  6  per  cent  solution  of  pure  para  phenylenediamine  hydro- 
chloride,  and  (2)  a  1.8  per  cent  solution  of  crystallised  guiacol 
in  96  per  cent  alcohol.  15  c.cms.  of  No.  1  are  added  to  135 
c.cms.  of  No.  2  and  the  mixture  preserved  in  an  amber-coloured 
bottle.  To  10  c.cms.  of  milk  add  0.5  c.cm.  of  the  reagent  and 
3  drops  of  hydrogen  peroxide  (3  per  cent).  A  blue  violet  colour- 
ation indicates  a  positive  peroxidase  reaction. 

Wilkinson  and  Peter's  Method.  To  10  c.cms.  of  milk  add 
1  c.cm.  of  a  10  per  cent  solution  of  benzidine  in  96  per  cent  alco- 
hol, 3  drops  of  30  per  cent  acetic  acid  and  finally  2  c.cms.  of 
3  per  cent  hydrogen  peroxide.  Peroxidases  produce  a  blue 
colouration  which  is  usually  localised  in  the  precipitated  casein- 
ogen. 

Bellei's  Method.  To  10  c.cms.  of  milk,  add  three  drops  of  a 
1.5  per  cent  aqueous  solution  of  ortol  and  two  drops  of  a  3 
per  cent  hydrogen  peroxide  solution.  A  red  colouration  indi- 
cates the  presence  of  peroxidases. 

Peroxidases,  like  reductase,  are  more  concentrated  in  the 
cream  layer  of  milk  though  it  is  impossible  to  establish  any 
definite  parallelism  between  the  butter  fat  content  and  the 
density  of  the  peroxidase  reaction. 

Catalase.  The  activity  of  this  ferment  is  estimated  by 
mixing  15  c.cms.  of  milk  and  5  c.cms.  of  2  per  cent  hydrogen 
peroxide  in  a  special  tube  devised  for  this  purpose  by  Lobeck. 
In  this  apparatus  the  oxygen  liberated  is  collected  and  measured 
in  a  graduated  tube  previously  filled  with  water.  The  libera- 
tion of  the  oxygen  is  accelerated  by  incubation  at  blood  heat 
for  two  hours.  Fresh  milk  usually  evolves  one  to  three  cubic 
centimetres  of  oxygen  and  results  materially  higher  than  these 
are  usually  indicative  either  of  excessive  bacterial  contamina- 
tion or  of  excessive  amounts  of  cellular  elements  produced  by 
physiological  or  pathological  irritations  of  the  udder. 

Amylase.     Into  each  of  10  test  tubes,  10  c.cms.  of  milk  are 


92  CHEMICAL  EXAMINATION 

placed  and  to  these  are  added  0.1,  0.2,  0.3  up  to  1  c.cm.  of  a  1 
per  cent  solution  of  soluble  starch  prepared  by  boiling  with  dis- 
tilled water  and  cooling.  After  shaking,  the  tubes  are  placed 
in  a  bath  at  43°-^5°  C.  for  one  hour  and  then  rapidly  cooled. 
To  each  is  added  1  c.cm.  of  a  solution  of  iodine  in  potassium 
iodide  (1  grm.  iodine,  and  2  grms.  potassium  iodide  in  300  c.cms. 
of  water),  and  the  colour  noted  immediately  after  shaking. 
The  recording  of  the  tints  admits  of  no  delay,  as  the  colours 
rapidly  fade  and  all  the  tubes  may  regain  their  original  shades. 
A  yellow  tint  indicates  total  conversion  of  the  starch  to  sugar, 
and  a  blue  one  unchanged  starch:  the  correct  reading  is  where 
the  yellow  just  commences  to  take  on  a  greyish  tint.  With 
normal  fresh  milk  this  will  usually  be  found  between  the  third 
and  fifth  tubes.  The  indications  of  this  test  are  similar  to 
those  of  the  catalase  test,  both  being  based  on  the  quantity  of 
1  cellular  elements. 

Galactase.  The  Babcock  and  Russell  method  is  probably 
the  most  reliable  for  the  estimation  of  this  ferment,  but  the  time 
required  for  its  execution  is  so  long  that  it  is  never  carried  out 
in  routine  examinations.  The  milk  is  incubated  at  blood  heat 
for  53  days  with  the  addition  of  sufficient  thymol  to  prevent 
bacterial  development  and  an  estimation  of  the  soluble  nitro- 
gen then  made.  The  difference  between  this  result  and  that 
originally  present  indicates  the  amount  produced  by  the  enzyme 
activity.  This  is  usually  less  than  1  per  cent  per  day. 

BIBLIOGRAPHY 

1.  Lythgoe.     Jour.  Ind.  and  Eng.  Chem.,     1914,     6,     906. 

2.  Leonard.     Analyst.     1896,  21,  157. 

3.  Richmond.     Dairy  Chemistry.     London,  1914,  186. 

4.  Shrewsbury  and  Knapp.     Analyst.     1909,  34,  12. 

5.  Thomson.     Analyst.     1903,  28,  184. 

6.  Stoke.    Analyst.     1897,  22,  320. 


CHAPTER  IV 
BACTERIA  IN  MILK 

MILK,  like  other  secretions,  is  sterile  at  the  moment  of 
secretion  but  it  is  usually  impossible  to  obtain  it  from  the  udders 
of  cows  in  this  condition  even  though  every  precaution  be 
taken  and  all  operations  are  conducted  under  strictly  aseptic 
conditions.  Many  have  held  that  bacteria  may  be  trans- 
ferred to  milk  directly  from  the  blood  stream  of  healthy  cows, 
but  this  view  is  now  generally  regarded  as  erroneous. 

Amongst  the  earliest  investigators  to  doubt  the  sterility  of 
the  udder  were  Bailey  and  Hall1  who  concluded  from  their 
experiments  that  the  milk  cistern  might  be  the  seat  of  bac- 
terial development  and  one  source  of  bacterial  contamination 
of  milk.  Ward2  carefully  examined  the  udders  of  19  milch 
cows  from  5  dairies  and  found  that  although  the  animals  were 
tubercular,  the  udders  were  normal.  He  found  that  all  the 
lactiferous  ducts  of  the  cows  were  contaminated  throughout 
with  bacteria  of  which  the  majority  were  cocci.  From  his 
studies  on  the  anatomy  of  the  udder  Ward  concluded  that 
with  the  possible  exception  of  the  sphincter  muscle,  at  the  lower 
end  of  the  teat,  no  obstruction  capable  of  excluding  bacteria 
from  the  milk  cistern  exists.  This  would  indicate  that  the 
source  of  contamination  of  milk  even  in  the  udder  is  external 
and  that  the  portal  of  entry  is  the  teat. 

Henderson 3  examined  a  number  of  cultures  from  seven 
normal  udders  and  obtained  growth  in  76  per  cent,  but  two 
cases  of  unexpanded  udders  from  heifers  gave  sterile  cultures 
from  the  milk  cistern,  ducts,  and  parenchyma. 

The  intra-mammary  contamination  of  milk  in  healthy  udders 
is  usually  small,  and,  although  in  some  exceptional  cases  counts 

93 


94  BACTERIA  IN  MILK 

as  high  as  15,000  per  c.cm.  have  been  obtained,  it  is  probable 
that  at  least  a  portion  of  this  number  was  due  to  external  con- 
tamination caused  by  faulty  aseptic  conditions  of  milk  with- 
drawal. 

Sedgwick  and  Batchelder4  found  that  with  moderate  pre- 
cautions on  the  part  of  the  milker,  the  organisms  in  fresh  milk 
may  not  exceed  500  to  1000  per  c.cm.,  but  if  ordinary  flaring 
pails  were  used  with  more  or  less  disturbance  of  the  bedding 
and  shaking  of  the  udder,  the  count  may  be  30,000  or  even  more. 

Park5  found  the  average  count  from  six  separate  cows, 
five  hours  after  collection,  to  be  4000  per  c.cm.  (minimum  400 
per  c.cm.)  and  the  average  of  25  cows  as  4550. 

McConkey6  observed  that,  with  ordinary  care  and  cleanli- 
ness, it  was  possible  to  obtain  milk  containing  less  than  1500 
bacteria  per  c.cm.  and  that  such  milk  should  not  contain  gas 
formers  in  less  than  50  c.cms. 

Von  Freudenreich 7  thought  it  would  be  easy  to  obtain 
sterile  milk  by  using  strict  asepsis  but  soon  found  otherwise. 
Such  milk  invariably  contained  250-300  bacteria  per  c.cm. 
though  the  hands  of  the  milkers  and  the  teats  of  the  cows  were 
washed  with  soft  soap  and  sterile  water,  then  with  servatol  soap 
and  sterile  water,  and,  finally  with  sterile  water  and  then  dried 
on  a  sterile  towel.  The  milkers'  hands  were  smeared  with  lano- 
line  and  the  fore  milk  rejected.  The  bacterial  content  of  the 
mixed  milk  of  28  cows  so  milked  varied  from  65-680  per  c.cm. 
Von  Freudenreich  and  Thoni  8  from  a  further  series  of  experi- 
ments concluded  that  freshly  drawn  milk,  even  when  every 
precaution  is  taken  against  contamination,  always  contains 
bacteria;  they  found  that  these  were  mostly  cocci  and  were 
derived  from  the  udder.  A  summary  of  the  more  important 
attempts  to  obtain  sterile  milk  is  as  follows : 

Von  Freudenreich,  200-300  per  c.cm. 

Szasz,  2  samples  sterile.     Average  of  11  =2700  per  c.cm. 

Hesse,  1600  per  c.cm. 

Marshall,  295  per  c.cm. 

Lux,  0  to  6800  per  c.cm. 


BACTERIAL  FLORA  OF  INTRA-MAMMARY  MILK        95 

Kolle,  80  to  15,000  per  c.cm. 

33  per  cent  less  than  300. 

50  per  cent  less  than  500. 

4. 7  per  cent  700-800 
Willem  and  Minne,  1  to  5  per  c.cm. 
Willem  and  Miele,    0  to  37  and  4  to  218  per  c.cm. 

Siebald,  (1)  Without    protective    measures.    Under    10    to 

several  thousands. 

(2)  After  soaping  the  udder.     0  to  85  per  c.cm. 

(3)  After  soaping  the  udder  and  disinfecting  with 

alcohol    and    milking    through    sterile     tubes, 
0  to  12  per  c.cm. 

All  these  numerous  experiments  prove  conclusively  that  some 
intra-mammary  contamination  of  milk  exists  and  it  will  be 
advisable  next  to  consider  the  nature  of  this. 

Like  Ward  2  Freudenreich  9  found  that  udder  contamination 
in  healthy  cows  was  mostly  caused  by  cocci,  but  Str.  lacticus 
(Heinemann)  was  only  found  in  three  cases  out  of  a  total  of 
fifteen.  B.  coli  was  never  found.  The  organisms  found  by 
Henderson  3  were  streptococci,  staphylococci  and  pseudo  diph- 
theria and  similar  results  were  obtained  by  Bergey.10 
/<"  From  these  and  other  results  it  would  appear  that  cocci, 
some  of  a  proteolytic  nature,  form  the  prevailing  type  found  in 
udders  and  that  the  lactic  acid  producing  bacteria,  both  coli- 
form  and  Str.  lacticus,  are  usually  absent.  Some  of  the  strep- 
tococci and  staphylococci  found  in  milk  produced  under  strictly 
aseptic  conditions  are  biochemically  similar  to  those  usually 
associated  with  inflammatory  processes  but  are  commonly  of 
much  lower  virulence. 

Experiments  on  the  viability  of  various  organisms  in  the 
environment  of  milk  ducts  has  shown  that  they  rapidly  die, 
many  bacteria  disappearing  within  a  few  days.  Savage n 
inoculated  the  teats  of  goats  with  streptococci  of  both  bovine 
and  human  origin  and  found  that  the  infecting  organism 
usually  died  in  a  few  weeks,  although  in  one  case  the  strep- 
tococci persisted  for  over  seven  months.  The  streptococci 
from  human  sources  were  usually  less  viable. 


96  BACTERIA  IN  MILK 

Although  the  majority  of  the  evidence  available  favours  the 
hypothesis  that  the  source  of  intramammary  contamination 
is  external  it  is  difficult  to  establish  this  entirely  on  account  of 
the  impossibility  of  putting  the  ducts  and  cisterns  in  a  sterile 
condition.  Once  infection  of  the  udder  has  occurred,  the 
organism,  finding  the  mammary  secretion  an  excellent  pabulum 
for  development,  persists  and  the  small  quantity  of  milk  re- 
maining from  one  milking  contaminates  the  next,  the  process 
being  repeated  until  the  cow  becomes  dry.  That  the  amount 
of  milk  allowed  to  remain  in  the  udder  has  a  very  material 
influence  upon  the  bacterial  count  of  the  milk  obtained  at  the 
next  milking  is  shown  by  the  experiments  of  Stocking,12  who 
found  as  the  average  of  ten  experiments  6542  bacteria  per  c.cm. 
in  milk  obtained  after  thoroughly  stripping  the  udder  as  against 
11,324  per  c.cm.  when  this  was  neglected.  The  importance  of 
this  factor  is  now  well  recognised  in  large  dairies  using  milking 
machines,  for  it  is  invariably  the  custom  to  take  out  the  last 
strippings  by  hand,  owing  to  the  impossibility  of  obtaining 
this  milk  by  means  of  the  machine.  This  hand-milked  secretion 
often  contains  more  bacteria  than  the  portion  immediately 
preceding  it,  due,  Stocking  suggests,  to  more  vigorous  manip- 
ulation of  the  udder  dislodging  bacteria  from  the  ducts  and 
which  remained  there  during  the  earlier  part  of  the  milking. 
The  contaminated  milk  left  in  the  ducts  is,  of  course,  mostly 
discharged  in  the  fore  milk  and  a  decreasing  count  is  obtained 
as  milking  proceeds.  Stocking  12  reports  the  following  results 
in  this  connection  as  the  averages  of  four  experiments : 

Bacteria  per  c.cm. 

Streams  1  and  2 10,143 

Streams  5  and  6 2,347 

Streams  9  and  10 272 

Streams  13  and  14'. 382 

Strippings 204 

The  influence  of  the  rejection  of  the  contaminated  fore 
milk  was  shown  by  the  following  figures: 


SOURCES  OF  BACTERIA  IN  MILK 


97 


BACTERIA  PER  C.CM. 

Total. 

Acid. 

Liquefying. 

Fore  milk  rejected 

499 
522 

99 

189 

33 

9 

Fore  milk  retained                       . 

Backhaus  13  reports  10,400  bacteria  per  c.cm.  in  fore  milk 
as  against  practically  sterile  strippings  whilst  the  author  in  one 
instance  obtained  50,000  per  c.cm.  in  the  fore  milk,  4000  in 
the  middle  milk  and  500  in  the  strippings.  The  advantage 
obtained  by  the  rejection  of  the  fore  milk  is  usually  much 
greater  than  is  indicated  by  Stocking's  results  reported  above, 
but  this  factor  is  largely  determined  by  the  precautions  observed 
in  other  directions  and  may  be  but  a  mirror  one  if  the  udders 
are  thoroughly  stripped  and  kept  clean  between  and  during 
milking  operations.  This  so-called  intramammary  contam- 
ination, which  is  really  external  contamination,  though  con- 
veyed to  the  milk  whilst  in  the  udder,  is,  however,  only  a  frac- 
tion of  the  external  contamination  that  reaches  the  milk  directly; 
this  is  especially  true  of  ordinary  market  milk.  The  external 
contamination  increases  at  every  stage  between  milking  and 
delivery  to  the  consumer  and  is  very  diverse  in  character. 
The  chief  sources  of  contamination  are : 


(1)  During  milking.        Bacteria  from 'dirty  udders,  flanks,  and  hands  of 

milkers:    also  aerial   contamination  with  dust 
of  food  or  litter. 

(2)  During  handling.      Dirty  containers,  strainers  and  cooling  apparatus. 

The  influence  of  bodily  cleanliness  of  the  cow  on  the 
bacterial  count  of  the  milk  obtained  has  been  investigated 
on  several  occasions.  Backhaus  13  found  20,600  bacteria  per 
c.cm.  in  the  milk  of  brushed  cows  as  against  170,000  per 
c.cm.  from  unbrushed  cows.  Stocking 12  reports  the  following 
results : 


BACTERIA  IN  MILK 


BACTERIA  PER  C.CM. 

Total. 

Acid. 

Liquefying. 

Brushed  

2268 
1207 

381 
213 

117 
59 

Unbrushed             

Wiping  the  udders  with  a  damp  cloth  previous  to  milking 
reduced  the  bacterial  count  from  7,058  to  716  per  c.cm.  Sim- 
ilar results  are  also  reported  by  Harrison.14  Orr  15  exposed 
plates  of  nutrient  medium  for  two  minutes  during  milking  and 
afterwards  incubated  them  for  four  days  at  20°  C.  The  results 
are  given  in  Table  XXXVIII. 

TABLE  XXXVIII 


Housing  of  the  Cows. 

Conditions  of  the  Cows. 

No.  of 
Experi- 
ments. 

Average 
Count  per 
Plate. 

Summer,  all  cows  out  .  . 

Untouched 

7 

440 

Summer,  all  cows  out  .  . 

Udders  and  flanks  washed 

and  brushed 

3 

170 

Winter,  cows  indoors.  .  . 

Untouched 

3 

4752 

Winter,  cows  indoors.  .  . 

Udders  and  flanks  brushed 

but  not  washed 

3 

1752 

Winter,  cows  indoors.  .  . 

Udders  and  flanks  brushed 

and    washed    and    left 

moist 

6 

230 

Winter,  cows  indoors.  .  . 

Udders  and  flanks  brushed, 

washed  and  dried 

3 

444 

The  practice  of  moistening  the  hands  of  the  milkers  by  the 
first  milk  streams  was  shown  by  Backhaus  to  increase  the  bac- 
terial count  from  5600  to  9000  per  c.cm.  The  effect  of  the 
character  of  the  litter  and  the  food  employed  is  very  marked 
as  is  also  that  of  the  influence  of  time  of  feeding.  The  ten- 
dency of  the  litter  to  dust  formation  is  a  factor  in  this  direction. 


EFFECT  OF  LITTER  AND  FEED 

TABLE  XXXIX 
BACTERIA  IN  LITTER  (BACKHAUS) 


99 


Litter. 

Organisms,  Per  Gram. 

Peat                                                        .    . 

2,000,000 

Good  straw  

7,500,000 

Bad  straw                             

10,000,000 

The  milk  obtained  contained 

Bacteria  per  C.cm. 

With  peat  litter 3500 

With  straw  litter 7330 

Backhaus  also  found  that  oil  cake  averaged  450,000  bacteria 
per  gram  and  bran  1,362,000  per  gram,  and  there  is  no  doubt 
that  other  dry  foods  also  contain  similar  large  numbers  of 
organisms.  Moist  foods  such  as  ensilage  would  have  no  effect 
if  entirely  consumed  but  would  be  equally  objectionable  as 
other  foods  if  allowed  to  dry. 

Stocking 12  reports  the  following  results  in  connection  with 
experiments  on  the  influence  of  feeding  before  and  after  milking. 

HAT  AND  CORN 


Total. 

Acid. 

Liquefying. 

Given  after  milking  

2096 

790 

108 

Given  before  milking  

3506 

1320 

196 

DRY  CORN 


Total. 

Acid. 

Liquefying. 

Given  after  milking  

1233 

297 

118 

Given  before  milking  

3656 

692 

123 

100  BACTERIA  IN  MILK 

The  results  of  Harrison 14  are  equally  interesting.  The 
organisms  falling  on  an  area  equal  to  a  circle  having  a  diameter 
of  12  inches  were  found  to  vary  from  12,210  to  42,750  during 
bedding,  feeding  and  cleaning  up,  whilst  one  hour  later  similar 
tests  gave  only  483  to  2370  organisms. 

Orr 15  by  exposing  plates  of  nutrient  medium  for  five  min- 
utes and  afterwards  incubating  for  four  days  at  20°  C.  obtained 
from  1260  to  4500  organisms  per  113  square  inches  (area  of 
circle  12  inches  in  diameter).  The  author  has  found  that  in 
clean,  well-ventilated  cow  byres  as  low  a  germ  content  as  200 
per  113  square  inches  could  be  attained  when  tested  with  plates 
of  nutrient  agar  for  five  minutes  and  incubated  at  37°  C.  for 
forty-eight  hours.  Coliform  bacilli,  as  shown  by  neutral  red 
lactose  agar  plates,  were  usually  absent. 

The  influence  of  milk  containers  is  also  well  marked.  Back- 
haus  found  that  fresh  milk  which  originally  contained  only  6600 
bacteria  per  c.cm.  was  increased  in  germ  content  to  97,000  per 
c.cm.  by  passage  through  six  containers.  Wooden  pails  were 
the  most  objectionable  in  this  respect  as  they  averaged  280,000 
germs  as  against  1690  for  galvanized  iron  and  1105  for  enam- 
elled ware.  Pails  after  rinsing  contained  28,600  organisms  and 
sterilized  pails  only  1300.  Harrison 14  also  investigated  the 
cleansing  of  cans;  by  rinsing  the  vessels  with  100  c.cms.  of 
sterile  water  he  obtained  the  following  results: 

BACTERIA  PER  C.CM. 

Improperly  cleaned  cans. .' 215,000-806,320 

Washed  with  tepid  water  and  scalding 13,080-  93,400 

Washed  with  tepid  water  and  steaming  5  mins. ..          355-     1,792 

Cloth  and  absorbent  cotton  strainers  may  also  be  a  source 
of  bacterial  contamination  unless  proper  precautions  are  taken. 

Milk  coolers  of  the  open  type  may  introduce  contamination 
from  both  the  cooler  itself  and  from  the  air.  This  is  well  exem- 
plified by  the  results  both  of  Orr 15  and  the  author.  (Table  XL.) 

Two  other  sources  of  milk  contamination  are  water  and  cow 
faeces.  It  is  obvious  that  all  the  water  used  for  cleansing  and 


COOLERS  •. 

TABLE  XL 

EFFECT  OF  MILK  COOLERS 
AVERAGE  OF  FOUR  EXPERIMENTS  (ORR) 


101 


BACTEHIA  PER  C.CM.  IN  MILK. 

Agar  48  Hrs. 
at  37°  C. 

Gelatine  96  Hrs. 
at  20°  C. 

Before  cooling  

26,000 
48,000 

39,000 

104,000 

After  cooling.                           .          ... 

Author's  results: 
Before  cooling. 

25,000 
400,000 

28,000 
30,000 

Coliform. 
4 
3,500 

2 

8 

After  cooling  

After  thorough  cleansing  of  coolers: 
Before  cooling  
After  cooling. 

rinsing  the  various  utensils  that  come  in  contact  with  the  milk  at 
various  stages  cannot  all  be  sterilised,  so  that  milk  will  contain 
a  number  of  the  bacteria  usually  found  in  water  supplies. 

Cow  faeces  may  also  be  conveyed  to  milk  by  falling  into 
milking  pails  after  becoming  dried  upon  the  udders  and  flanks 
of  the  cows.  This  danger  may  be  eliminated  as  has  previously 
been  pointed  out  by  washing  these  portions  of  the  beasts. 
Savage  16  gives  several  analyses  of  fresh  cow  excreta.  (Table 
XLL) 

From  this  general  consideration  of  the  various  sources  of 
milk  contamination  it  is  obvious  that  milk  even  whilst  fresh 
may  contain  large  numbers  of  an  almost  infinite  variety  of 
organisms.  Before  taking  up  the  methods  of  examination  for 
these  organisms  it  will  be  advisable  to  consider  the  effect  of 
storage,  for  milk  samples  are  rarely  taken  of  the  product  in  a 
fresh  condition.  This  point  is  also  important  in  considering 
the  conditions  requisite  for  preventing  bacterial  multiplication 


102 


IN  MILK 

TABLE  XLI 
BACTERIA  IN  COW  FAECES  (SAVAGE) 


ORGANISMS  PER  GRAM. 


Source. 

B.  coli. 

Streptococci. 

B.  enteritiditis 
sporogenes 
Spores. 

Cow  No.  1 
2 
3 
4 

100,000-  1,000,000 
1,000-        10,000 
1,000,000-10,000,000 
1,000,000-10,000,000 

10,000-    100,000 
100,000-1,000,000 
More  than  10,000,000 
100,000-1,000,000 

100-1000 
10-  100 
10-  100 
100-1000 

in  the  interval  that  elapses  between  sampling  and  the  labora- 
tory examination. 

Park 17  took  two  samples  of  milk,  one  containing  3000  organ- 
isms per  c.cm.  (agar  forty-eight  hours  at  37°  C.)  and  the  other 
30,000  per  c.cm.  and  stored  portions  at  various  temperatures. 
After  various  intervals  of  time  the  bacterial  counts  were  again 
taken  with  the  results  shown  in  Table  XLII. 

The  author  has  made  similar  tests  but,  in  addition  to  the 
total  bacterial  count,  an  estimation  was  made  of  the  B.  coli 
group  by  plating  on  rebipelagar  (neutral  red  bile  salt  agar)  and 
incubating  at  37°  C.  for  twenty-four  hours.  The  total  bacteria 
were  counted  on  +1.0  per  cent  nutrient  agar  after  forty-eight 
hours  incubation  at  37°  C. 

It  will  be  noticed  in  both  these  series  of  experiments,  and 
especially  in  Park's,  that  at  the  lower  temperature  there  is  at 
first  an  apparent  diminution  in  the  total  bacterial  count  and 
that  this  phenomenon  is  more  definite  and  more  prolonged  at 
the  lowest  temperature  used.  These  observations  have  been 
confirmed  by  many  experimenters  and  led  to  the  hypothesis 
that  milk  possessed  a  weak,  though  definite  bactericidal  action : 
this  is  usually  referred  to  as  the  germicidal  action  of  milk. 
M.  J.  Rosenau  18  thoroughly  investigated  this  phenomenon  and 
concluded  that  no  true  germicidal  action  took  place,  but  that 


EFFECT  OF  TEMPERATURE 


103 


TABLE  XLII 

Upper  figures  represent  sample  No.  1.     Original  count    3,000. 
Lower       "  "       No.  2.  "  "     30,000. 


Temperatures, 
°F. 

TIME  WHICH  ELAPSED  BEFORE  MAKING  TEST. 

24  Hours. 

48  Hours. 

96  Hours. 

168  Hours. 

32 

2,400 
30,000 

2,100 
27,000 

1,850 

24,000 

1,400 

19,900 

39 

2,500 
38,000 

3,600 
56,000 

218,000 
4,300,000 

4,200,000 
38,000,000 

42 

2,600 
43,000 

3,500 
210,000 

500,000 
5,760,000 

46 

3,100 
42,000 

12,000 
360,000 

50 

11,600 
89,000 

540,000 
1,940,000 

55 

18,800 
187,000 

3,400,000 
38,000,000 

60 

180,000 
900,000 

28,000,000 
168,000,000 

68 

450,000 
4,000,000 

25,000,000,000 
25,000,000,000 

86 

1,400,000,000 
14,000,000,000 

94 

25,000,000',000 
25,000,000,000 

fresh  milk  appeared  to  act  as  a  weak  antiseptic.  Vigorous 
shaking  of  the  samples  demonstrated1  that .  the  reduction  in 
count  was  more  apparent  than  real  and  suggested  that  the 


104 


BACTERIA  IN  MILK 


= 


o 


CO   O 

+?  II 

03  ^ 


So 


»-i          (N          <N 
>O 


:-2 


38383831383 

r°     —'     r°      — '     P         -      ,°         -'     -O  '     -O 


GERMICIDAL  ACTION  105 

organisms  had  aggregated  into  clusters  under  the  influence  of 
agglutinins.  The  so-called  germicidal  action  was  also  found  to 
be  specific  but  the  specificity  of  different  samples  was  variable. 
Further  proof  of  the  fact  that  this  phenomenon  must  be  attrib- 
uted to  agglutinins  rather  than  to  bacteriolysins  was  found  in 
the  behaviour  of  heated  milk.  Heating  to  56°  C.  for  thirty 
minutes,  a  condition  which  destroys  bacteriolysins,  weakens 
but  does  not  entirely  inhibit  the  action;  it  is  entirely  destroyed 
at  75°  C. 

St.  John  and  Pennington  19  found  that  milk,  after  heating 
to  79°  C.  for  twenty  minutes,  not  only  failed  to  show  an  ap- 
parent diminution  in  the  number  of  organisms  but  also  showed 
a  much  greater  rate  of  bacterial  development  throughout  the 
period  of  observation.  They  point  out  that  this  is  a  serious 
objection  to  pasteurisation  as  a  reinfected  heated  product 
exerts  no  restraining  effect  upon  the  invading  organisms  and 
may,  therefore,  be  more  infective  than  raw  milk  receiving  the 
same  original  contamination. 

Stocking,20  who  investigated  this  question,  concluded  that 
the  apparent  diminution  of  organisms  capable  of  development 
on  solid  media  was  really  due  to  bacteria  finding  the  milk  a 
pabulum  to  which  they  are  unaccustomed  and  consequently 
died  at  a  faster  rate  than  they  could  multiply;  he  found  that 
this  resting  stage  was  scarcely  observable  with  common  lactic 
acid  organisms  which  appeared  to  develop  more  or  less  rapidly 
and  continuously  from  the  moment  of  their  introduction  into 
the  milk.  The  absence  of  a  "  germicidal  effect  "  with  common 
lactic  acid  organisms  was  confirmed  by  Rosenau  and  others  and 
supports  rather  than  impairs  the  validity  of  the  agglutination 
hypothesis  by  accentuating  its  specificity.  The  resting  stage 
pointed  out  by  Stocking  must  also  be  a  factor,  but  cannot 
wholly  account  for  it  as  it  fails  to  explain  the  comparative 
absence  of  the  phenomenon  in  heated  milk  unless  it  is  assumed 
that  heating  has  resulted  in  chemical  changes  that  have  pro- 
duced a  more  favourable  environment  for  bacterial  develop- 
ment. Once  this  resting  period  or  germicidal  phase  has  passed, 


106  BACTERIA  IN  MILK 

bacterial  development  sets  in,  the  rapidity  of  which  depends 
upon  the  temperature  at  which  the  sample  is  stored.  The 
organisms  that  have  gained  admittance  to  the  milk  do  not  all 
find  that  substance  a  suitable  medium  for  reproduction,  but 
certain  classes  develop  rapidly  and  ultimately  one  or  more  of 
these  classes  predominates.  The  bacteria  that  reproduce  most 
rapidly  may  be  roughly  divided  into  three  groups  according  to 
their  biochemical  characteristics,  viz.,  acid  producers,  pro- 
teolytic,  and  inert  organisms.  Ayers  and  Johnson21  made  a 
fourth  general  division  by  separating  the  alkali  producers,  but 
this  group  is  usually  included  in  the  inert  group.  The  classifi- 
cation was  based  upon  the  behaviour  of  the  organisms  on  litmus 
lactose  gelatine,  the  acid  producers  being  those  capable  of 
producing  red  colonies,  the^  proteolytic  being  liquefiers,  and  the 
balance,  having  no  well-defined  characteristics  on  this  medium, 
the  inert  group.  The  acid  producers  may  be  subdivided  into 
two  further  groups  according  to  their  ability  to  ferment  lactose 
with  the  production  of  gas.  This  separates  the  coliform  organ- 
isms, which  produce  hydrogen  and  carbon  dioxide  from  lac- 
tose in  addition  to  lactic  acid,  and  the  ordinary  lactic  acid 
organisms  which  do  not  give  any  gaseous  products. 

Although  different  samples  of  milk  will  all  show  varying 
rates  of  development  of  the  various  groups,  a  general  dis- 
cussion of  this  point  will,  perhaps,  be  facilitated  by  consider- 
ation of  a  concrete  example.  Table  XLIV  shows  the  results 
of  a  daily  examination  of  a  sample  of  milk  kept  comparatively 
cool. 

All  three  groups,  in  this  example,  developed  rapidly,  the 
greatest  relative  increase  being  shown  by  the  coliform  organ- 
isms, until  a  maximum  was  reached  at  the  end  of  five  days.  At 
this  stage  the  acidity  was  44°  and  this  amount  was  evidently 
sufficient  either  alone  or  in  conjunction  with  the  other  products 
of  metabolism,  to  restrain  the  rate  of  production.  The  coli- 
form organisms  were  the  first  to  be  affected,  although  the  other 
acid  producers  and  to  an  even  smaller  degree,  the  liquefiers, 
were  restrained.  On  the  tenth  day  the  liquefiers  commenced 


BACTERIAL  DEVELOPMENT  IN  MILK 


107 


x     § 


« 

t3 


, 
1-8 


IO   >O   CO 


CO          £ 
00  O5 


888     888 

o  o  o       o  o  o 


CO    ^2    C^  CD    GO    ^D    ^O    ^^    *O 


o  o  o  >o  o 
cT  o"  o"  cT  o~ 
co  *o  t>  co  t~ 


s 


1 

^ 


8 


V  V 


T-H     T^     (N     i-H 


OOOOOOOOOOOOGOOOOOOOOOO 
iOOiOiOrli^iOTtH^^T^T^T^O 


108  BACTERIA  IN  MILK 

to  gradually  decrease  and  a  few  days  later  it  was  impossible 
to  make  an  accurate  estimation  of  their  number  owing  to  the 
overgrowth  of  acid  producers.  The  inert  group  developed 
well  during  the  first  period  and,  after  a  reduction  at  the  tenth 
day  period,  persisted  to  the  end  of  the  experiment.  The  sample 
ultimately  developed  a  prolific  growth  of  torulse. 

In  considering  the  relative  development  of  various  groups 
in  milk,  due  regard  must  always  be  given  to  the  two  important 
factors,  viz.,  temperature  and  initial  content,  that  determine 
the  results. 

The  effect  of  temperature  was  carefully  investigated  by 
Conn  and  Esten,22  who  plated  out  practically  fresh  milk  usually 
containing  20,000  bacteria  per  c.cm.  on  litmus  lactose  agar  and 
found  that  they  were  able  to  distinguish  no  less  than  15  different 
groups  merely  by  their  macroscopic  appearance.  They  made 
two  series  of  experiments,  the  first  at  37°  C.,  20°  C.,  and  10°  C. 
and  the  second  at  20°  C.,  10°  C.,  and  1°  C.  The  plating  inter- 
vals were: 

37°  C.  at  2  hour  intervals 

20°  C.  at  6  hour  intervals 

10°  C.  at  12  hour  intervals 
1°  C.  at  1  day  intervals. 

The  main  conclusions,  as  summarised  by  Conn  and  Esten, 
were: 

(1)  The  effect  of  variations  of  temperature  upon  the  devel- 
opment of  different  species  of  bacteria  in  milk  is  not  always  the 
same  under  apparently  identical  conditions.     In  spite  of  such 
variations,   there   seems   to   be   clearly   discernible   a   normal 
development  of   bacteria  associated  with  different   tempera- 
tures. 

(2)  There  is,  in  all  cases,  a  certain  period  at  the  beginning 
when  there  is  no  increase  in  the  total  number  of  bacteria. 
During  this  period  some  species  are  multiplying  whilst  others 
are  apparently  dying.     The  length  of  this  period  depends  upon 
the  temperature.     At  37°  C.  it  is  very  short,  while  at  10°  C. 
it  may  last  from  six  to  eight  days,  since,  at  this  temperature, 


TEMPERATURE  AND  BACTERIAL  FLORA      109 

milk  may,  in  six  days,  actually  contain  fewer  bacteria  than  when 
fresh. 

(3)  After  this  preliminary  period,  there  always  follows  a 
multiplication  of  bacteria;    but  the  types  that  develop  differ 
so  markedly,  that  samples  of  the  same  milk  kept  at  different 
temperatures  are,  at  later  periods,  very  different  in  their  bac- 
terial content,  even  though  they  contain  the  same  number  of 
bacteria. 

(4)  The  development  of  the  ordinary  lactic  species  Bact. 
lactis  acidi  (Str.  lacticus),  in  practically  all  cases  checks  the\ 
growth  of  other  species  of  bacteria,  and,  finally,  kills  them,  < 
since  the  bacteria  regularly  decrease  in  actual  numbers  after ' 
the  lactic  bacteria  have  become  very  abundant. 

(5)  In  practically  all  samples  of  milk  kept  at  20°  C.,  the 
multiplication  of  the  Str.  lacticus  *  begins  quickly  and  pro- 
gresses with  great  rapidity.     They  grow  so  rapidly  that  they 
produce  acid  enough  to  curdle  the  milk  in  about  forty  hours, 
the  growth  of  other  species  being  held  in  check.     Milk  when 
curdled  at  this  temperature  shows  a  smooth  acid  curd,  with  no 
gas  bubbles. 

(6)  A  totally  different  result  appears  in  milk  kept  at  37°  C. 
The  results  are  somewhat  more  variable  than  at  20°  C.     Occa- 
sionally the  Str.  lacticus  grows  vigorously  at  this  temperature, 
but  the  common  result  is  a  development  of  the  B.  lactis  serogenes 
type.     It  forms  a  curd  full  of  gas  bubbles.     If  B.  coli  communis 
is  in  the  milk,  this  also  grows  luxuriantly  at  37°  C. 

(7)  In  milk  kept  at  10°  C.,  neither  of  the  types  of  bacteria 
seems  to  be  favoured.     The  delay  in  growth  lasts  two  to  three 
days,  after  which  all  types  of  bacteria  appear  to  develop  some- 
what   uniformly.     Sometimes    the    lactic    bacteria    develop 
abundantly,   sometimes  only  slightly.     The  neutral  bacteria 
always  grow  rapidly,  and  the  liquefiers  in  many  cases  become 
abundant.     In  time,  the  milk  is  apt  to  curdle,  commonly  with 


*  Str.  lacticus  has  been  substituted  for  B.  lactis  acidi  (Hueppe)  in 
order  to  avoid  confusion  with  B.  acidi  lactici  (Escherich). 


110  BACTERIA  IN  MILK 

an  acid  reaction,  but  it  never  shows  the  predominance  of  Str. 
lacticus  found  at  20°  C. 

(8)  From  our  experiments  there  seems  to  be  no  difference 
between  the  effect  of  10°  and  1°  upon  the  bacteria,  except  upon 
the  rapidity  of  growth.     1  °  C.  very  markedly  checks  the  growth 
of  bacteria;    but,  later  they  grow  in  large  numbers.     As  at 
10°  C.,  the  lactic  bacteria  fail  to  outgrow  the  other  species,  so 
that  all  types  develop  abundantly.     A  few  species  appear  to  be 
particularly  well  adapted  to  this  low  temperature  and  are  espe- 
cially abundant  at  the  end  of  the  experiment. 

(9)  The  curdling  point  appears  to  be  quite  independent  of 
the  number  of  bacteria  present.     In  one  sample  at  37°  C.,  the 
milk  curdled  with  only  8,000,000  organisms  per  c.cm.  while 
in  others  there  have  been  found  4,000,000,000  per  c.cm.  without 
any  curdling.     These  differences  are  apparently  due  to  the 
development  of  enzymes,  and  partly  to  the  products  of  some 
species  neutralising  the  action  of  others.     The  amount  of  acid 
present  at  the  time  of  ordinary  acid  curdling  does  not  widely 
vary. 

(10)  Milk  is  not  necessarily  wholesome  because  it  is  sweet, 
especially  if  it  has  been  kept  at  low  temperatures.     At  the 
temperature  of  an  ice  box  milk  may  remain  sweet  for  a  long 
time  and  yet  contain  enormous  numbers  of  bacteria,  among 
which  are  species  more  likely  to  be  unwholesome  than  those 
that  develop  at  20°  C. 

Although  these  results  show  that  temperature  exerts  a 
selective  action  on  the  bacterial  flora  it  must  not  be  forgotten 
that  this  may  be  wholly  or  partially  negatived  by  a  predominance 
of  any  particular  species  in  the  original  milk.  For  example, 
milk  produced  under  good  conditions  and  containing  less  than 
10,000  bacteria  per  c.cm.  will  very  rarely  show  a  predominance 
of  coliform  organisms  even  when  incubated  at  37°  C.  The 
curd  produced  by  this  class  of  milk  is  almost  invariably  of  the 
smooth  acid  type  produced  by  Str.  lacticus  and  seldom  gives 
the  gas-blown  curd  typical  of  the  B.  coli  group.  An  examina- 
tion of  the  type  of  curd  produced  on  incubation  at  37°  C.  has 


EFFECT  OF  LOW  TEMPERATURES 


Ill- 


been  suggested  as  a  simple  method  of  determining  the  pre- 
vailing type  of  organisms  and  will  be  considered  in  detail  on 
p.  197. 

The  development  of  bacteria  in  milk  at  low  temperatures 
was  especially  studied  by  Re  venal,  Hastings  and  Hammer.23 
Two  samples  of  milk  differing  widely  in  bacterial  content  were 
stored  at  0°  C.  and  the  count  made  at  intervals  on  lactose 
agar  by  incubating  at  37°  C. 

TABLE  XLV 


Age  of  Milk,  Days. 

Dairy  Milk. 

Barn  Milk. 

0 

130,000 

3,500 

6 

72,500 

4,050 

15 

633,500 

52,900 

20 

3,230,000 

1,240,000 

36' 

34,950,000 

4,800,000 

74 

91,500,000 

36,500,000 

106 

39,750,000 

192,500,000 

160 

32,650,000 

361,000,000 

That  profound  modifications  had  occurred  was  shown  by 
the  fact  that  at  the  end  of  the  experiment  over  70  per  cent  of 
the  caseinogen  was  digested.  The  total  nitrogen  decreased,  due 
to  liberation  of  nitrogen  in  the  free  state.  Pennington  24  also 
found  a  digestion  of  caseinogen  when  milk  was  stored  at  low 
temperatures,  over  50  per  cent  being  digested  in  five  to  six 
weeks  at  29°-32°  F. 

The  above  results  show  the  importance  of  storing,  milk  at 
as  low  a  temperature  as  is  practicable;  although  50°  F.  may  be 
regarded  as  the  critical  point  for  bacterial  development,  efforts 
should  be  made  to  lower  the  temperature  of  milk  samples  as 
far  as  possible  if  more  than  a  few  hours  (3-4)  elapse  between 
collection  and  examination.  If  the  samples  are  immediately 
surrounded  with  ice  they  may  be  kept  for  twenty-four  hours 
without  altering  the  significance  of  the  results  although  the 


112  BACTERIA  IN  MILK 

bacterial  count  may  vary  slightly;  the  direction  of  this  varia- 
tion will  depend  upon  the  condition  of  the  milk  when  sampled, 
low  counts  tending  to  decrease  and  high  counts  to  become  still 
Jiigher,  thus  leaving  the  general  significance  unaltered. 

BIBLIOGRAPHY 

1.  Bailey  and  Hall.     Centralbl.  f.  Bakt.     1895,  Abt.  2,  793. 

2.  Ward.     Bull.  178,  Cornell  Expt.  Sta.     1898. 

3.  Henderson.     J.  Roy.  San.  Inst.     1904,  25,  563. 

4.  Sedgwick  and  Batchelder.     Boston  Med.  Jour.     1892,  126,  25. 

5.  Park.     Jour,  of  Hyg.     1901,  1,  391. 

6.  McConkey.     Jour,  of  Hyg.     1906,  6,  385. 

7.  Von  Freudenreich.  '  Centralbl.  f.  Bakt.     1901,  Abt.  2,  8,  674. 

8.  Von  Freudenreich  and   Thoni.     Centralbl.  f.  Bakt.     1903,  Abt.  2, 

10,  305. 

9.  Von  Freudenreich.     Centralbl.  f.  Bakt.     1903,  401. 

10.  Bergy.     Bull.  125,  Penn.  Dept.  of  Agr.     1904. 

11.  Savage.     Milk  and  Public  Health.     London,  1915,  p.  19. 

12.  Stocking.     Rpt.  Storr's  Expt.  Agr.  Sta.     1906,  Bull.  42. 

13.  Backhaus.     Molkerei  Zeit.,  1898,  No.  4. 

14.  Harrison.     Rpt.  Ontario  Agr.  Dept.     1896,  109-113. 

15.  Orr.     Rpt.  on  Milk  Contamination,  1908. 

16.  Savage.     Bact.  Examination  of  Water  Supplies.     London,  1906,  p.  35 

17.  Park.     Jour,  of  Hyg.     1901,  1,  398. 

18.  Rosenau.     U.  S.  A.  Pub.  Health  and  Marine  Hosp.  Service,  Hyg. 

Lab.  Bull.  56. 

19.  St.  John  and  Pennington.     Jour.  Inf.  Dis.,  1907,  4,  647. 

20.  Stocking.     Storr's  Expt.  Agr.  Sta.  Bull.  28,  1904. 

21.  Ayres  and  Johnson.     U.  S.  A.  Dept.  of  Agr.,  Bull.  126.     1910. 

22.  Conn  and  Esten.  ,  Rpt.  Storrs  Expt.  Agr.  Sta.     1904,  27. 

23.  Ravenal,  Hastings,  and  Hammer.     Jour.  Inf.  Dis.,     1910,  7,  38. 

24.  Pennington.    J.  of  Bio.  Chem.     1908,  4,  353. 


CHAPTER  V 
THE  ENUMERATION  OF  BACTERIA  IN  MILK 

AN  ,  approximate  determination  of  the  total  bacteria  in 
milk  by  plating  on  solid  media  has,  for  many  years,  been  one 
usually  made  in  connection  with  the  examination  of  milk,  and, 
although  later  work  has  shown  that  the  number  so  obtained 
is  usually  but  a  small  fraction  of  the  total  number  present, 
these  methods  have  been  generally  retained  on  account  of  their 
convenience,  and  the  results  are  usually  described  as  the  total 
bacterial  counts.  There  has  been  considerable  difference  of 
opinion  amongst  sanitarians  regarding  the  value  of  this  test, 
for,  whilst  some  regard  the  total  number  of  minor  importance, 
others  believe  that  much  valuable  information  can  be  obtained 
by  this  determination  alone.  The  fact  that  the  great  majority 
of  regulations  for  the  sale  of  milk,  where  regulations  have  been 
enacted,  contain  no  other  clause  with  reference  to  bacteria 
than  a  maximum  number  clause,  is  sufficient  to  show  the  trend 
of  opinion  on  this  subject.  Those  who  deprecate  the  value  of 
the  total  bacteria  enumeration  take  the  stand  that  the  large 
majority  of  the  bacteria  usually  found  in  milk  are  harmless 
saprophytes,  and  that  their  determination  is  more  or  less  a 
waste  of  time  and  labour.  Whilst  the  former  statement  is 
undoubtedly  true,  the  latter  must  be  emphatically  denied. 
Until  bacteriological  technique  becomes  so  developed  that 
routine  methods  can  be  applied  for  the  detection  of  pathogenic 
organisms,  those  employed  in  milk  examination  must  be  con- 
tent with  the  inferential  tests  obtained  by  determination  of  the 
saprophytes.  As  has  been  shown  in  the  preceding  chapter, 
milk  drawn  with  reasonable  aseptic  precautions  from  the 
udders  of  cows  contains  but  few  bacteria,  and,  if  properly 

113 


114        THE  ENUMERATION  OF  BACTERIA  IN  MILK 

treated,  can  be  delivered  in  that  condition  to  the  consumer. 
Laxity  on  the  part  of  the  producer  or  dairyman  by  the  use  of 
dirty  containers  or  lack  of  cooling  facilities,  produces  conditions 
favourable  to  the  development  of  bacteria  for  which  milk  forms 
an  excellent  nidus.  Once  the  milk  has  become  contaminated, 
the  organisms  multiply  very  rapidly  under  favourable  con- 
ditions, and,  by  the  time  the  milk  reaches  the  consumer,  have 
become  excessive  in  number.  A  low  bacterial  count  is  an  "a 
posteriori "  argument  that  proper  and  reasonable  care  has 
been  exercised  in  the  production  of  the  sample  examined,  and  it 


TABLE  XL VI 
TOXICITY  OF  MILK  (DELEPINE) 

MIXED  MILK  COMING  MORE  THA:S   40  MILES  AND  GENERALLY  KEPT 

24-60  HOURS 


Mean  Temp,  in  Shade,  Manchester,  during  time  specimens 
were  kept,  Degrees  Fahrenheit. 

Percentage  of  Good 
Specimens. 

30-35 

58 

35-40 

38.5 

40-45 

40 

45-50 

20 

50-55 

55-60 

Nil 

MILK  FROM  SHORT  DISTANCES  (LESS  THAN  20  MILES)  USUALLY  KEPT 
LESS  THAN  10  HOURS 


Mean  Temp,  in  Shade,  Manchester,  during  time  specimens 
were  kept,  Degrees  Fahrenheit. 

Percentage  of  Food 
Specimens. 

50-55 

100 

55-60 

88.8 

60-65 

73.2 

65-70 

70-75 

50.0 

REASONS  FOR  DETERMINATION  OF  TOTAL  COUNT   115 

might  fairly  be  inferred  that  such  milk  is  less  likely  to  contain 
pathogenic  organisms  then  one  produced  by  men  of  careless 
and  slovenly  habits.  Farmers  who  take  a  pride  in  their  produce 
are  more  naturally  liable  to  prevent  infection  of  the  milk  by 
supervision  of  their  employees,  but  even  if  this  be  not  true,  it 
must  be  admitted  that  the  conditions  which  tend  to  keep  in 
check  the  saprophytes  also  tend  to  minimise  the  relative 
infectiveness,  so  that  to  this  extent  at  least,  must  credit  be 
given  to  careful  producers  and  dairymen.  Other  conditions 
being  equal,  the  total  bacterial  count  is  a  measure  of  relative 
infectiveness.  This  statement  is  supported  by  the  work  of 
Delepine  l  on  the  toxicity  of  the  Manchester  milk  supply.  He 
found  that  "  mixed  milk  .  .  .  showed  an  increase  of  virulence 
on  inoculation  into  guinea  pigs  in  proportion  to  the  mean 
temperature  in  the  shade  in  Manchester  during  the  time  the 
specimen  was  kept/'  The  results  are  given  in  Table  XLVI, 
all  tuberculous  specimens  being  excluded. 

Increased  temperature  and  keeping  period  result  in  an 
increased  count  so  that  the  above  statement  can  be  reduced  to 
one  stating  that  the  virulence  to  guinea  pigs  was  proportional 
to  the  bacterial  count.  Further  figures  reported  by  Delepine 
regarding  the  relative  toxicity  of  cooled  and  uncooled  milk 
confirm  this. 

TABLE  XLVII 
TOXICITY  OF  MILK 


No.  of  Samples. 

Percentage  of  Toxic 
Samples. 

1896-1897. 
1898-1901. 

Unref  rigerated  milk  .  .  . 
Refrigerated  milk  

141 

1782 

10.7 
2.1 

Delepine  states  that  "  the  difference  would  probably  have 
been  greater  if  the  milk  had  been  cooled  immediately  after 
milking." 


116        THE  ENUMERATION  OF  BACTERIA  IN  MILK 

Results  reported  by  the  Chicago  Department  of  Health2 
on  the  relative  toxicity  of  raw  and  pasteurised  milk  also  confirm 
this  hypothesis. 

After  this  consideration  of  the  "  raison  d'etre  "  of  the  bac- 
terial enumeration,  the  methods  by  which  this  is  accomplished 
will  now  be  treated  in  detail.  These  may  be  divided  into  two 
groups:  (a)  plating  methods  and  (6)  direct  microscopical 
methods.  The  former  are  based  upon  the  ability  of  the  indi- 
vidual organisms  to  reproduce  at  such  a  rate  upon  the  medium 
employed  as  to  produce  a  visible  colony  within  the  period  of 
incubation,  and  the  latter  upon  suitable  preparation  for  direct 
enumeration  under  high  magnification. 

Until  within  the  last  few  years  the  former  method  was  the  one 
usually  employed,  and  as  it  is  still  in  universal  use,  it  will  be 
convenient  to  discuss  it  first. 

Plain  nutrient  gelatine  prepared  with  fresh  beef  infusion 
was  first  used  with  the  plate  method  for  the  enumeration  of 
bacteria  in  milk  and  still  enjoys  considerable  repute  with  many 
workers  for  this  purpose,  the  colonies  being  usually  counted 
after  four  to  five  days  incubation  at  20°  to  22°  C.  In  late 
years,  however,  and  especially  in  America,  this  method  has 
largely  been  supplanted  by  the  substitution  of  agar  for  gelatine 
and  the  incubation  period  reduced  to  forty-eight  hours  at  blood 
heat.  Although  the  agar  medium  does  not  produce  as  many 
visible  colonies  within  the  incubation  period  as  the  gelatine  one, 
it  possesses  certain  advantages  which  more  than  offset  this 
drawback.  In  routine  work  it  is  very  desirable  that  results 
should  be  obtained  in  the  shortest  possible  time,  and  in  this 
respect  the  agar  medium  is  decidedly  preferable  as  it  reduces 
the  time  required  by  60  per  cent.  If  necessary  the  colonies 
may  be  counted  after  twenty-four  hours  incubation,  but  the 
results  so  obtained  do  not  exhibit  the  sharp  contrasts  given  by 
the  longer  period.  Some  of  the  author's  results  are  given  in 
Table  XLVIIL3 

The  average  of  the  ratio  of  the  forty-eight  hour  count  to  the 
twenty-four  hour  C9\int  is  3.4,  but  if  the  abnormal  value  of 


INCUBATION  PERIOD 


117 


TABLE  XLVIII 

EFFECT   OF   INCUBATION    PERIOD   ON 
STANDARD  AGAR 


MILK   COUNTS   ON 


Sample  No. 

INCUBATION  PERIOD  AT  37°  C. 

.    48  Hours 

24  Hours. 

48  Hours. 

24  Hours 

684 

64,000 

140,000 

2.2 

685 

1,500 

21,000 

14.0 

686 

55,000 

94,000 

1.7 

687 

11,600 

16,000 

1.4 

688 

8,500 

18,000 

2.1 

689 

44,000 

105,000 

2.4 

690 

500 

1,600 

3.2 

691 

20,000 

63,000 

3.1 

692 

2,300 

4,800 

2.1 

693 

2,500 

7,000 

2.8 

695 

11,000 

21,000 

1.9 

sample  685  is  omitted,  it  becomes  2.1  with  a  variation  of  from 
1.4  to  3.2.  Conn  4  reports  "  that  in  the  averages  in  28  series  of 
samples  submitted  to  four  laboratories,  the  forty-eight  hour 
count  was  the  larger  in  25  cases,  smaller  in  one  case,  and  the 

TABLE  XLIX 


BACTERIA  PER  C.CM.  ON 


Sample  No. 

Standard  Agar  48  Hours 
at  37°  C. 

Standard  Gelatine  5  Days 
at  20°  C. 

1 

123,000 

224,000 

2 

8,000 

8,600 

4 

10,300 

8,800 

5 

1,300,000 

1,500,000 

7 

85,000 

113,000 

8 

155,000 

240,000 

9 

12,700 

8,600 

118         THE  ENUMERATION  OF  BACTERIA  IN  MILK 


same  in  two  cases."  The  averages  of  the  whole  series  (omitting 
the  samples  counting  in  millions)  were  299,000  for  the  twenty- 
four  hour  count  and  147,000  for  the  twenty-four  hour  count. 
This  gives  a  ratio  of  2.03  :  1.  It  is  obvious  that  no  constant 
factor  can  be  employed  for  the  ratio  of  the  twenty-four  hour 
count  to  the  forty-eight  hour  count  as  this  will  vary  with  the 
bacterial  flora.  For  the  same  reason  the  results  obtained  with 
the  use  of  different  media  are  not  comparable  although  they 
usually  vary  in  the  same  direction.  This  is  well  illustrated  by 
the  results  given  in  Table  XLIX  which  shows  a  comparison 
between  standard  agar  and  gelatine. 

It  will  be  seen  that  when  the  bacterial  count  is  low,  the  dif- 
ference between  the  gelatine  and  agar  count  is  but  small, 
and,  although  the  gelatine  medium  usually  gives  the  higher 
result,  this  is  not  an  invariable  rule ;  the  agar  occasionally  gives 
a  higher  count,  but  this,  in  the  author's  experience,  only  occurs 
in  a  small  minority  of  cases  and  as  the  bacterial  count  increases, 
the  ratio  of  the  gelatine  count  to  the  agar  count  usually  becomes 
greater. 

That  the  addition  of  1  per  cent  of  lactose  to  both  nutrient 
gelatine  and  agar,  favours  more  rapid  reproduction  is  shown  in 
Table  L. 

TABLE  L 


Sample  No. 

Standard  Agar 
48  Hours 
at  37°  C. 

Lactose  Agar 
+  1  Per  Cent 
48  Hours. 

Standard 
Gelatine 
5  Days 

Lactose 
Gelatine, 
5  Days 

at  37°  C. 

at  20°  C. 

at  20°  C. 

1 

123,000 

180,000 

224,000 

240,000 

2 

8,000 

8,400 

8,600 

8,300 

3 

12,000 

11,000 

6,500 

12,300 

4 

10,300 

11,900 

8,800 

13,500 

5 

1,300,000 

1,350,000 

1,500,000 

1,850,000 

6 

600,000 

60,000 

65,000 

84,000 

7 

85,000 

140,000 

113,000 

156,000 

8 

155,000 

230,000 

240,000 

500,000 

9 

12,700 

12,800 

8,600 

14,000 

EFFECT  OF  SUGAR  IN  MEDIA  119 

Heinemann  and  Glenn  5  investigated  the  action  of  dextrose 
and  lactose-litmus  agar  at  20°  C.  and  37°  C.  and  concluded 
that  incubation  at  20°  C.  for  three  days  was  the  most  preferable 
technique  as  this  temperature  is  less  selective  in  its  action  than 
higher  ones  and  so  yields  more  information  as  to  the  original 
flora.  After  twenty-four  hours  incubation  they  found  the  37° 
count  to  be  the  higher,  but  this  was  reversed  after  a  further 
twenty-four  hours  incubation  and  the  difference  was  still  more 
marked  after  seventy-two  hours.  Dextrose  and  lactose  litmus 
agar  gave  but  insignificant  differences  in  the  total  count  but 
the  former  showed  a  decidedly  higher  percentage  of  acid  col- 
onies, due,  it  is  suggested,  to  colonies  of  the  B.  aerogenes  type 
becoming  red  only  temporarily  and  finally  assuming  a  blue 
colour.  For  this  reason  Heinemann  and  Glenn  prefer  dex- 
trose to  lactose.  The  high  counts  obtained  by  these  observers 
seem  to  indicate  that  the  samples  had  been  kept  for  some  time 
and  that  considerable  reproduction  had  taken  place.  This 
possibly  had  an  effect  on  the  results  obtained.  For  example : 
Str.  lacticus,  which  is  usually  abundant  in  stale  milk,  grows  well 
at  20°,  but  at  37°  produces  colonies  in  forty-eight  hours  that 
are  barely  visible  even  with  the  aid  of  a  low-power  magnifying 
glass  and  are  usually  overlooked  when  the  medium  is  tinted  with 
litmus. 

The  Committee  on  Methods  of  Milk  Analysis  appointed  by 
the  American  Public  Health  Association  to  investigate  the 
various  details  of  the  plate  method  using  an  agar  medium 
reported  as  follows  (Am.  J.  of  Pub.  Hyg.,  18,  431). 

Acidity  (to  phenolphthalein  at  boiling  point).  Of  the 
acidities +0.5,  +1.0,  +1.5  and  2.0,  an  acidity  of  +1.5  per  cent 
gave  the  best  results. 

Lactose.  0,  1,  2,  3,  and  4  per  cent  of  lactose  was  tried 
at  incubation  temperatures  of  20°  C.  and  37°  C.  At  37°  C., 
they  found  that  the  medium  free  from  lactose  was  preferable, 
but  at  20°  C.  the  one  containing  1  per  cent  of  sugar  was  the 
best. 

Whey,  Plain,  and  4  Per  Cent  Lactose  Agar  media  were  com- 


120         THE  ENUMERATION  OF  BACTERIA  IN  MILK 

pared  in  74  tests.  In  28  tests  ordinary  agar  gave  the  best  re- 
sults, whey  agar  in  24  tests,  and  lactose  agar  in  22  tests.  They 
found  that  whey  agar  favoured  the  growth  of  lactic  acid  organ- 
isms and  ordinary  agar  of  organisms  other  than  lactic  acid 
producers. 

Agar  and  Gelatine.  Litmus  lactose  agar  at  37°  C.  was  com- 
pared with  litmus  lactose  gelatine  at  20°  C.  in  25  tests:  of  these 
gelatine  gave  higher  results  in  18  tests  and  agar  in  7.  Where 
gelatine  showed  the  higher  count  the  percentage  difference  was 
much  greater  than  where  agar  showed  the  higher  numbers.  It 
was  also  found  that  the  differentiation  of  species  was  much 
better  on  gelatine  but  that  there  was  a  considerable  loss  of 
plates  with  this  medium. 

Both  media  were  used  at  20°  C.  in  24  tests  and  in  this  series 
gelatine  was  the  better  in  14  and  agar  in  10  samples.  When 
beef  peptone  gelatine  at  20°  C.  with  seventy-two  hours  incu- 
bation was  tried  against  beef  peptone  agar  at  37°  C.  with 
twenty-four  hours  incubation,  gelatine  gave  the  higher  count 
in  18  tests,  agar  in  4  tests,  and  in  one  test  they  gave  identical 
results.  The  total  gelatine  count,  however,  was  more  than 
double  that  on  the  agar  plates.  The  standard  method  for  the 
examination  of  milk  as  adopted  by  the  American  Public  Health 
Association  in  1912  was  the  plate  method  with  a  plain  agar 
medium  of  +1.5  per  cent  acidity,  made  with  beef  infusion 
and  1  per  cent  each  of  peptone  and  dried  agar.  The  1916 
report  recommended  certain  alterations;  concentrated  beef 
extract,  3  gms.  per  litre,  was  substituted  for  beef  infusion  and 
the  acidity  was  reduced  to  +1.0  per  cent:  the  quantity  of 
peptone  was  reduced  to  5  gms.  per  litre  and  the  agar  in- 
creased to  1.2  per  cent  of  the  dried  material.  Although  the 
author  has  not  compared  fresh  beef  infusion  media  with  similar 
media  prepared  with  Lemco  for  the  enumeration  of  bacteria  in 
milk,  his  experience  with  water  was  that  the  Lemco  media  in- 
variably gave  higher  and  more  consistent  results.  The  reason 
for  variable  results  with  beef  infusions  or  decoctions  lies  in  the 
difficulty  in  obtaining  solutions  of  even  approximately  con- 


ACIDITY  OF  MEDIUM 


121 


stant    composition    and    in    the    variable    quantity  of  alkali 
required  for  the  adjustment  of  the  acidity. 

Clark  3  has  pointed  out  that  the  method  of  adjusting  the 
acidity  of  media,  as  recommended  in  the  standard  methods  of 
analysis,  is  not  scientific  in  principle  and  that  it  does  not  ensure 
a  constant  hydrogen  ion  concentration.  Various  batches  of 
media  prepared  by  different  workers  and  adjusted  to  an  acidity 
of  +1  per  cent  by  the  standard  method  (titration  of  the  boiling 
medium  with  alkali  using  phenolphthalein)  were  found  to  have 
very  different  H  ion  potentials  when  tested  by  the  electrical 
method.  No  results  are  given  by  Clark  as  to  the  effect  of  this 
variation  on  the  bacterial  reproduction  in  these  media  but  the 
comparative  experiments  of  a  group  of  New  York  bacteriolo- 
gists indicate  that  any  variation  due  to  this  cause  is  insignificant 
and  can  safely  be  ignored.  In  these  experiments  media  were 
prepared  by  four  laboratories  and  supplied  to  Dr.  Conn,  of 
Middletown,  Conn.,  who  plated  out  two  samples  of  milk  on 
each  medium  in  triplicate.  The  results  were  as  follows: 


Medium. 

Borden. 

North. 

Board  of  Health. 

Lederle. 

Sample  1  ... 
Sample  2  ... 

12,000 

305,000 

15,000 
290,000 

14,000 

280,000 

13,000 
279,000 

Three  of  the  above  media  gave  an  acidity  of  +1.0  per  cent, 
as  determined  by  Conn,  and  the  fourth  +0.9  per  cent.  These 
results  show  that  media  prepared  in  various  laboratories  accord- 
ing to  standard  methods  give  results  as  close  as  can  be  expected 
from  a  consideration  of  the  technique. 

The  technique  of  bacterial  enumeration  in  milk  was  care- 
fully investigated  by  the  New  York  group  of  bacteriologists 
above  referred  to  and  the  results  summarised  by  Conn.4 
Samples  of  various  grades  of  milk  and  cream  were  prepared  by 
Conn  and  duplicate  samples  forwarded  to  the  various  laborator- 
ies partaking  in  the  work.  As  the  samples  invariably  included 
duplicate  samples  under  different  numbers,  each  sample  was 


122         THE  ENUMERATION  OF  BACTERIA  IN  MILK 

not  only  examined  in  four  laboratories  but  each  laboratory 
was  unknowingly  checking  the  accuracy  of  its  own  work. 
The  various  points  investigated  were  as  follows: 

1.  Method  of  Inoculation.  Three  methods  were  employed: 
(a)  Measurement  of  the  sample  into  plates  and  pouring  the 
agar  from  flasks,  (6)  measurement  into  plates  but  pouring  the 
agar  from  tubes,  and  (c)  inoculation  of  the  tubes  and  pouring 
into  plates  after  rolling.  The  results  obtained  show  the  slight 
superiority  of  the  tube  inoculation  method  but  the  advantage 
is  so  slight  as  to  be  of  no  real  importance.  In  the  few  cases 
where  methods  (a)  and  (6)  were  compared,  (a)  gave  higher 
results  though  there  is  no  manifest  reason  why  this  should 
occur.  In  laboratories  where  large  numbers  of  samples  are 
examined  the  slight  superiority  of  the  tube  inoculation  method 
is  more  than  offset  by  the  economy  in  material  and  labour 
effected  by  the  use  of  the  flask  method.  The  author's  experi- 
ence has  been  that,  although  the  time  required  for  plating  sam- 
ples was  not  very  much  reduced,  the  preparation  of  media  was 
greatly  facilitated  and  the  cost  reduced. 

Composition  of  Media.  In  one  series  three  different  media 
were  used  (a)  standard  agar  (beef  bouillon  with  the  addition  of 
1  per  cent  agar  and  peptone  and  adjusted  to  +1.5  per  cent 
acidity),  (6)  standard  agar  with  the  substitution  of  Liebig's 
extract  for  beef  infusion,  and  (c)  agar  prepared  with  beef 
extract  but  containing  only  one-twelfth  the  quantity  in  (6)  and 
having  an  acidity  of  +0.3  per  cent. 

The  results  showed  that 

In  30  samples  (a)  medium  gave  the  highest  count. 
In  27  samples  (c)  medium  gave  the  highest  count. 
In  20  samples  (6)  medium  gave  the  highest  count. 

So  far  as  the  actual  numbers  were  concerned  the  differences 
were  of  no  real  significance  so  that,  in  this  respect,  the  media 
were  of  equal  value.  The  size  of  the  colonies  on  (c)  medium 
was  generally  small  and  rendered  accurate  counting  more  dif- 
ficult. Against  this  disadvantage  must  be  placed  the  decreased 


ACCURACY  OF  COUNTS  123 

trouble  experienced  with  spreaders.  Observations  for  spreaders 
indicated  that  128  were  found  with  (a)  medium,  21  with  (6) 
medium  and  23  with  (c)  medium.  On  the  whole,  it  would 
appear  that  the  (6)  medium  was  the  most  satisfactory. 

Uniformity  of  Technique.  The  several  series  of  compara- 
tive examinations  produced  some  interesting  data  on  the  influ- 
ence of  technique.  In  the  first  series  when  each  laboratory 
used  the  technique  as  previously  developed  in  that  laboratory, 
the  results  on  duplicate  samples  showed  a  variation  factor  of 
from  1.3  to  43.2  with  an  average  of  6.2.  The  variation  factor 
was  obtained  by  dividing  the  highest  result  by  the  lowest. 
Duplicate  analyses  in  each  laboratory  also  showed  variations, 
the  average  factors  varying  from  2.1  to  4.8  with  a  general 
average  of  3.7. 

In  a  second  series  of  tests  the  various  laboratories  all  em- 
ployed identical  technique  as  to  shaking  of  sample,  diluting, 
pipetting,  inoculating,  and  counting  of  plates.  As  it  was  found 
in  the  first  series  that  one  laboratory  employed  a  magnifying 
lens  for  counting  plates  and  another  the  naked  eye,  it  was 
decided  to  use  a  standard  lens  in  all  laboratories  and  to  deter- 
mine the  personal  error  in  counting  by  an  exchange  of  incubated 
plates.  The  results  showed  that  the  personal  error  may  be  a 
serious  one,  for,  although  the  variation  in  duplicate  counts  of 
identical  plates  was  usually  small,  the  extreme  variation  was 
nearly  100  per  cent.  In  this  series  the  average  variation  in 
each  laboratory  was  from  1.6  to  2.2  with  a  general  average 
of  1.8. 

A  five-day  count  was  also  compared  with  the  two-day  count 
and,  although  the  results  were  usually  higher  they  were  not 
uniformly  so.  There  seems  to  be  no  apparent  advantage  attain- 
able by  prolonging  the  incubation  period  beyond  the  usual 
forty-eight  hour  period. 

In  the  third  series  the  effect  of  agitation,  amongst  other 
points,  was  determined,  and  although  the  results  are  not  con- 
clusive they  indicate  the  importance  of  standardising  this  por- 
tion of  the  technique.  In  the  third  and  fourth  series  the  plate 


124          THE  ENUMERATION  OF  BACTERIA  IN  MILK 

method  of  enumeration  was  also  compared  with  the  direct 
microscopical  method  of  Breed  but  this  will  be  dealt  with  later. 

From  a  consideration  of  this  work  Conn  pointed  out  that 
variations  in  technique  are  much  more  important  than  the  com- 
position of  the  medium,  and  that  variations  in  results  may 
reasonably  be  expected,  even  under  the  best  conditions  due 
(1)  to  clumping  of  the  bacteria,  and  (2)  to  the  bacteria  being 
in  non-uniform  suspension  and  not  in  solution.  These  two 
factors  render  it  improbable  that  two  small  samples  will  contain 
equal  numbers  of  organisms,  and  the  lower  the  total  number  of 
bacteria  the  greater  will  this  divergence  become.  Conn  ex- 
pressed the  opinion  that  "  individual  counts  under  the  best 
conditions  are  subject  to  considerable  variation  and  that  no 
single  individual  count  can  be  relied  upon."  ..."  It  is  not 
possible  to  rely  upon  a  greater  accuracy  than  100  per  cent  even 
when  the  average  of  more  than  one  sample  is  obtained,  although 
most  of  the  results  fall  considerably  below  this  limit." 

During  1915  the  author  made  a  series  of  duplicate  examina- 
tions of  milk  by  plating  one  of  the  routine  samples  in  duplicate 
daily;  in  this  series  plates  containing  ytnr  c.cm.  and  TWO  c.cm. 
were  inoculated  and  counted  with  a  low-power  glass  after  forty- 
eight  hours  incubation  at  37°  C.  Porous  covers  were  used  to 
prevent  loss  of  plates  by  spreaders.  In  142  samples  the  differ- 
ence between  duplicate  determinations  varied  from  zero  to 
464  per  cent  with  an  average  variation  of  24.7  per  cent.  Ex- 
pressed as 'a  variation  factor  the  average  was  1.25  (1.247)  with  a 
maximum  of  4.64.  The  bacterial  count  varied  from  1600  per 
c.cm.  to  1,200,000  per  c.cm.  and  it  was  with  the  best  grade  milks, 
i.e.,  those  containing  less  than  10,000  per  c.cm.,  that  the  vari- 
ations were  the  largest.  This  was  anticipated  from  a  consider- 
ation of  the  frequency  distribution  in  the  largest  amount  of 
sample  plated  and  could  have  been  reduced  by  inoculating 
larger  quantities.  This  was  not  done  because  the  labour  in- 
volved in  so  treating  all  samples,  when  but  very  few  were  of 
this  grade,  was  not  justified  by  the  increased  precision  so  ob- 
tainable, for  whether  a  sample  contains  1600  or  5000  organisms 


AMERICAN  STANDARD  METHODS  125 

per  c.cm.  has  no  real  bearing  on  its  hygienic  quality.  This 
series  of  comparative  results  is  not  so  important  as  that  reported 
by  Conn  because  of  the  psychological  factor;  both  the  person 
plating  out  the  samples  (A.  J.  S.)  and  the  one  counting  the 
plates  (J.  R.)  were  aware  that  these  determinations  were  being 
made,  and  although  every  endeavour  was  made  to  honestly 
record  the  actual  conditions  found,  it  is  recognised  that  the 
results  are  subject  to  these  limitations. 

The  detailed  technique  for  the  plate  method  as  adopted  by 
the  American  Public  Health  Association  in  1916  is  as  follows: 

Dilutions.  For  samples  of  unknown  character  dilutions 
of  1  to  100,  1  to  1000,  1  to  10,000  shall  be  made,  using  sterile 
water  and  pipettes  after  the  ordinary  method.  In  case  the 
character  of  the  milk  is  known,  less  than  three  dilutions  may  be 
made ;  but  in  no  case  shall  less  than  two  plates  for  each  sample 
be  made.  Grade  A,*  or  its  equivalent,  should  be  plated  in 
duplicate,  and  a  dilution  lower  than  1  to  100  may  be  used. 

Shaking.  Samples  must  be  shaken  twenty-five  times. 
Shaking  is  defined  as  meaning  a.  rapid  up  and  down  motion 
with  an  excursion  of  not  less  than  1  foot. 

Pipettes.  Pipettes  must  be  made  to  deliver  between  grad- 
uation marks,  not  simply  to  deliver. 

Pouring  Plates.  The  melted  agar  must  be  poured  promptly 
after  measuring  out  the  proper  quantities  of  milk.  Not  more 
than  twelve  plates  must  be  allowed  to  accumulate  after  the 
distribution  of  the  milk  into  the  plates  before  pouring  the 
agar. 

Incubation  and  Counting.  One  standard  temperature  only 
is  recognised — forty-eight  hour  incubation  at  37°  C. 

If  possible  count  those  plates  containing  between  30  and 
200  colonies.  If  there  are  none  such,  count  those  plates  con- 
taining nearest  to  200  colonies.  The  whole  number  of  colonies 
on  the  plate  shall  be  counted  where  the  plates  contain  less  than 
200  colonies. 

*  Milk  usually  containing  less  than  10,000  bacteria  per  c.cm. 


126          THE  ENUMERATION  OF  BACTERIA  IN  MILK 

Counting  Lens.  The  lens  recommended  by  the  Committee 
in  1914  is  more  fully  defined.  It  is  known  as  Engraver's  lens 
No.  146,  Bausch  &  Lomb  catalogue.  It  is  designated  as  3JX, 
its  magnification  being  2|  diameters.  Persons  who  are  near- 
sighted should  wear  their  ordinary  glasses  while  using  this  lens. 
Farsighted  persons  should  use  the  lens  without  their  glasses. 

Direct  Methods.  The  direct  methods  of  enumeration  of 
bacteria  in  milk  are  of  comparatively  recent  development;  in 
these  the  milk  or  centrifugalised  sediment  is  smeared  over  a 
slide,  and,  after  suitable  staining,  examined  under  a  high- 
power  objective  and  the  bacteria  counted.  The  direct  method 
as  modified  by  Slack  8  is  as  follows.  Two  c.cms.  of  the  sample, 
after  thorough  shaking,  are  inserted  into  special  tubes  with 
rubber  stoppers  at  each  end,  and  centrifugalised  for  ten  minutes 
at  2500  revolutions  per  minute  in  a  special  apparatus.  This 
apparatus  is  a  modification  of  the  one  used  by  Stewart  of  Phil- 
adelphia for  leucocyte  estimation,  and  consists  of  an  aluminium 
disc  and  cover  10  inches  in  diameter  and  f  inch  in  depth,  fitted 
to  hold  twenty  tubes  arranged  radially.  This  apparatus  is 
manufactured  by  the  International  Instrument  Co.,  of  Cam- 
bridge, Mass.,  and  can  be  used  with  the  usual  electrical  cen- 
trifuge. After  centrifugalising,  the  tubes  are  carefully  removed, 
and,  to  obtain  the  sediment  with  the  least  disturbance,  the  tube 
is  held  with  the  cream  end  downwards,  whilst  the  cream  layer 
is  removed  by  means  of  a  platinum  loop.  The  milk  is  then 
carefully  poured  out  without  permitting  air  bubbles  to  ascend 
the  tube,  and  finally,  with  the  tube  in  the  same  position,  the 
other  stopper  is  removed  and  the  sediment  is  smeared  on  a  glass 
slide  with  the  aid  of  a  drop  of  sterile  water.  An  area  of  459 
cms.  is  a  convenient  one  and  squares  of  this  size  may  be  marked 
off  on  a  strip  of  glass  with  a  blue  grease  pencil.  The  smear  is 
dried,  fixed  by  heat,  and  stained  with  methylene  blue.  The 
specimen  is  then  examined  under  a  A  inch  oil  immersion  lens 
and  the  organisms  counted.  Each  coccus,  bacillus,  diplococcus, 
or  chain  represents  a  colony  on  the  1-10,000  plate  of  the  same 
sample  when  grown  on  agar  for  twenty-four  hours  at  37°  C. 


DIRECT  METHODS  127 

This  factor  of  10,000  was  modified  later  to  20,000  in  order  to 
correspond  to  the  forty-eight  hour  incubation  period.  Whilst 
it  was  not  claimed  that  the  whole  of  the  bacteria  are  contained 
in  the  sediment,  it  was  asserted  that  in  99  per  cent  of  the  sam- 
ples a  representative  number  is  so  precipitated,  and  that  this 
number  bears  a  fairly  constant  relation  to  the  bacterial  count  as 
determined  by  plating  on  agar.9 

Slack,  in  a  series  of  over  2200  samples,  compared  the  results 
obtained  by  the  centrifuge  and  plate  methods  (twenty-four 
hours  at  37°  C.)  and  an  error  of  less  than  1  per  cent  was  made  in 
passing  as  below  500,000  bacteria  to  the  cubic  centimetre, 
milks  which  the  plates  showed  to  be  above  this  limit. 

This  method  has  also  been  examined  by  Gooderich 10 
who  reports  very  favourably  upon  it  and  remarks  that  very  little 
improvement  can  be  made  upon  the  factor  2X104  (20,000)  for 
converting  the  microscopical  results  to  the  forty-eight  hour 
count  on  agar.  He  reports  the  limits  for  the  factor  as  being 
from  0.66  X104  to  6.0  X104.  With  a  standard  of  50,000  bac- 
teria per  c.cm.  -he  found  that  the  direct  method  wrongly  passed 
8.6  per  cent,  and  wrongly  condemned  8.9  per  cent,  but  that 
when  the  standard  was  raised  to  100,000  these  figures  were 
reduced  to  1.4  and  4.3  per  cent,  respectively.  In  considering 
these  results  it  is  important  to  note  that  all  the  determinations 
were  made  on  samples  secured  from  the  University  Stock  Farm. 
The  variations  in  bacterial  content  of  such  samples  would  not 
be  nearly  so  great  as  is  met  with  in  routine  work  on  various 
market  milks  of  unknown  origin,  with  the  consequence  that  the 
errors  would  be  minimised.  The  small  variation  in  the  counts 
is  clearly  indicated  by  the  fact  of  the  mention  of  only  a  1-1000 
dilution  being  used  for  plating.  Such  a  procedure  is  impossible 
in  routine  work  on  market  samples  in  which  the  count  may  vary 
from  a  few  hundreds  to  5,000,000  or  even  more.  In  view  of 
the  excellent  results  obtained  by  Gooderich,  the  writer  experi- 
mented with  this  method,  although  a  consideration  of  the  fun- 
damental principles  did  not  lead  to  an  anticipation  of  a  high 
degree  of  accuracy  3.  If  the  results  were  to  correspond  with  the 


128         THE  ENUMERATION  OF  BACTERIA  IN  MILK 

usual  plate  count  it  was  essential  that  a  constant  proportion  of 
the  bacteria  capable  of  development  on  agar  in  forty-eight  hours 
at  37°  C.  must  be  precipitated  during  the  process  of  centri- 
fugalisation.  A  portion  of  the  bacterial  flora  of  milk,  however, 
does  not  produce  visible  colonies  on  agar  under  the  usual  condi- 
tions, so  that  either  these  organisms  must  remain  in  suspension 
or  the  error  due  to  them  be  counterbalanced  by  some  other 
factor. 

No  difficulty  was  found  with  the  technique  until  the  micro- 
scopical examination  was  made.  The  representative  field  in 
which  the  organisms  were  to  be  counted  was  difficult  to  find 
owing  to  the  widely  differing  content  of  various  fields.  In 
order  to  minimise  this  source  of  error  ten  fields  were  taken  at 
random  and  the  average  calculated. 

In  a  series  of  market  samples,  for  which  the  standard  was 
500,000  bacteria  per  c.cm.  not  a  single  sample  was  condemned 
which  passed  the  plate  method;  on  the  other  hand,  17  per  cent 
were  passed  which  were  condemned  by  the  plate  method. 
According  to  these  results  the  direct  method  outlined  above 
would  not  be  oppressive  on  the  milk  producer,  and  its  adoption 
would  be  tantamount  to  lowering  the  standard.  In  this  series 
the  factor  (c)  for  the  conversion  of  microscopic  counts  to  plate 
counts  varied  within  very  wide  limits,  viz.,  from  0.4 X104  to 
33. OX  104,  and  the  author  is  convinced  that  this  is  largely  due  to 
the  difficulty  found  in  obtaining  an  even  distribution  of  organ- 
isms on  the  slide.  Two  observers  obtained  widely  varying 
results  from  the  same  slide;  a  condition  fatal  to  accuracy. 
Breed,11  in  1911,  improved  this  method  by  making  a  direct 
smear  of  the  milk  and  thus  eliminating  the  centrifuge  with  its 
many  unknown  factors.  Breed's  method  consists  essentially 
in  spreading  a  small  volume  of  milk  over  a  marked  area  and 
examining  under  a  high-power  objective  after  washing  out  the 
fat  followed  by  suitable  staining.  Skar,12  in  1912,  independ- 
ently developed  a  similar  method  which  differs  only  in  the 
manner  of  staining  and  in  allowing  the  fat  to  remain  in  the 
smears.  Rosam's  method 13  differs  essentially  from  Skar's 


DIRECT  METHODS  129 

method  only  in  the  method  of  smear  examination:  these  are 
made  on  a  cover  glass  and  examined  whilst  wet. 

In  some  of  the  comparative  experimental  work  reported  by 
Conn  and  discussed  on  page  123,  a  series  of  bacterial  counts 
was  made  by  Breed  and  this  was  supplemented  in  a  further 
series  by  the  inclusion  of  Brew,  a  co-worker  with  Breed.  These 
experimenters  made  microscopical  counts  on  the  samples  plated 
by  other  observers,  and  Conn  14  considered  that  when  the 
groups  of  organisms  only  were  counted,  the  count  agreed  some- 
what closely  with  the  plate  count.  When  raw  market  milk 
was  examined,  the  variations  found  were  generally  not  greater 
than  the  differences  between  the  plate  counts  in  various  labora- 
tories, but  for  high-grade  raw  milk  and  pasteurised  products  it 
is  comparatively  useless.  The  details  of  Breed's  process  are 
as  follows:  0.01  c.cm.  of  milk,  from  a  well-shaken  sample,  is 
measured  out  by  means  of  an  accurately  calibrated  special 
pipette  and  deposited  on  a  glass  slide  on  which  an  area  of  1 
square  centimetre  has  been  previously  marked  out.  The  drop  is 
evenly  smeared  over  this  area  with  a  stiff  needle  and  gently 
dried  at  about  50°  C.  The  slide  is  then  placed  in  a  Coplin 
staining  jar  containing  xylol  or  gasoline  to  remove  the  fat,  and, 
after  drying,  fix,ed  in  alcohol  (70  to  95  per  cent).  Immediately 
afterwards  the  smear  is  stained  with  1  per  cent  aqueous  methy- 
lene  blue  and,  finally  decolourised  to  a  light  blue  in  95  per  cent 
alcohol.  The  microscopical  examination  is  made  with  a  ^ 
inch  oil  immersion  objective.  In  order  to  find  the  factor  for 
converting  the  number  of  organisms  per  field  into  organisms 
per  cubic  centimetre  the  diameter  of  the  field  is  determined  with 
a  stage  micrometer.  The  factor  is  then  calculated  from  the 
formula: 


where  y  is  the  factor  sought,  x,  the  area  of  the  smear  in  square 
millimetres,  and  R  the  radius  of  the  field. 

In  practice  it  is  convenient  to  pull  out  the  draw  tube  until 


130          THE  ENUMERATION  OF  BACTERIA  IN  MILK 

the  area  of  the  field  is  of  such  a  value  as  will  give  a  value  to 
y  having  as  many  ciphers  as  possible.  The  following  are  the 
most  satisfactory. 

When  R  =  0.080  m.m.,  y  =  500,000 
When  R  =  0.089  m.m.,  y  =  400,000 
When  R  =  0.101  m.m.,  y  =  300,000 

When  the  desired  result  is  obtained  the  position  of  the  draw 
tube  is  noted  and  always  set  at  this  point  in  future  examinations. 
In  order  to  get  results  comparable  with  the  plate  method,  only 
the  groups  or  clumps,  together  with  isolated  bacilli  are  counted ; 
individual  cocci,  diplococcus  or  streptococcus  chains,  and  rod 
forms  where  the  plane  of  division  shows  clearly,  are  counted  as 
individuals.  The  number  of  fields  to  be  examined  must  be 
determined  by  the  frequency  of  the  organisms.  It  is  obvious 
that  with  a  factor  of  300,000  to  500,000,  this  method  is  of  the 
greatest  advantage  when  the  count  averages  one  clump  or  more 
per  field;  with  high-grade  milks  under  10,000  bacteria  per 
c.cm.  the  number  of  fields  to  be  examined  would  be  so  large, 
if  reasonable  precision  is  to  be  obtained,  as  to  consume  as  much 
time  as  the  plate  method.  Dead  bacteria  are  counted  with  the 
living,  so  that  this  process  is  not  applicable  to  pasteurised 
products;  it  would,  however,  be  of  advantage  in  determining 
the  quaKty  before  pasteurisation.  A  collateral  advantage  of 
this  method  is  that  in  addition  to  the  quantitative  estimation  of 
the  bacteria,  a  cell  count  can  be  made  at  the  same  time  and 
information  obtained  regarding  the  bacterial  flora. 

As  an  indirect  method  for  estimating  the  number  of  bacteria, 
Barthol,15  in  1908,  suggested  the  employment  of  methylene 
blue.  It  was  found  by  Barthol  and  confirmed  later  by  Jensen 
and  Muller,  that  the  time  required  to  decolourise  methylene 
blue  bears  a  relationship  to  the  number  of  bacteria  present. 
Fred  16  showed  that  21  of  23  species  of  milk  bacteria  were  capa- 
ble of  reducing  methylene  blue  and  that  each  species  has  a 


INDIRECT  METHODS 


131 


different  coefficient  of  velocity;  the  velocity  of  reduction  was  a 
linear  function  of  the  temperature  (up  to  37°  C.)  and,  finally, 
ceased  with  exhaustion  of  the  medium.  It  was  formerly  sug- 
gested that  the  reduction  of  methylene  blue  in  this  "  slow 
reductase  test  "  as  it  is  usually  termed,  was  due  to  enzymes 
present  in  the  intramammary  milk,  but  it  is  now  generally  held 
that  such  milk  does  not  contain  reducing  substances  and  that 
the  reduction  is  due  to  intra  and  extra  cellular  products  of  bac- 
terial origin. 

Fred  17  in  an  examination  of  200  samples  of  milk  by  this 
method  (adding  1  c.cm.  of  a  0.05  per  cent  solution  of  pure 
methylene  blue  in  0.4  per  cent  saline  to  10  c.cms.  of  milk  and 
holding  at  40°  Q.)  found  that  the  time  required  for  reduction 
was  proportional  to  the  bacterial  count.  His  figures  are  given 
in  Table  LI,  each  group  representing  the  average  of  20  samples. 


TABLE  LI 


Group  Number. 

Average  Number  of  Bacteria 
per  c.cm. 

Average  Time  of  Reduction 
in  Hours. 

1 

29,647 

11.9 

2 

73,587 

9.7 

3 

160,150 

9.5 

4 

283,250 

8.0 

5 

548,300 

7.8 

6 

1,016,600 

4.7 

7 

1,469,650 

3.1 

8 

2,505,000 

27 

9 

4,690,000 

1.5 

10 

8,624,800 

1.0 

Barthol 18  found  that  samples  containing  more  than  10,000,- 
000  bacteria  per  c.cm.  and  50  per  cent  of  those  containing 
4-10  millions  per  c.cm.  reduced  within  one  hour.  He  concluded 
that  10  millions  per  c.cm.  was  the  lowest  limit  that  could  be 
estimated  by  this  method  and  that  below  this  limit  there  is  no 


132         THE  ENUMERATION  OF  BACTERIA  IN  MILK 

relationship  between  the  number  of  bacteria  and  the  time 
required  for  decolourisation. 

The  author  examined  a  number  of  milks  by  this  test  in  1914 
but  was  unable  to  find  any  merit  in  it.  Almost  all  the  samples 
failed  to  decolourise  in  the  six  hours  that  were  available  for 
observation  under  ordinary  laboratory  conditions,  and  they  had 
generally  showed  reduction  by  the  following  morning  (twenty- 
one  hours).  As  over  90  per  cent  of  these  samples  contained 
less  than  one  million  bacteria  per  cubic  centimetre  these  results 
are  not  inconsistent  with  Fred's  (vide  supra),  but  as  the  time 
of  reduction  could  only  be  determined  within  wide  limits  no 
real  information  could  be  deduced  as  to  the  bacterial  condi- 
tion of  the  sample,  except  that  it  did  not  contain  very  excessive 
numbers.  Samples  that  were  allowed  to  stand  and  develop 
large  numbers  of  organisms  showed  small  reduction  periods  and 
it  would  seem  that  it  is  in  the  detection  of  such  milk  that  the 
chief  value  of  the  test  lies. 

A  further  rapid  indirect  method  that  has  been  suggested 
for  the  approximate  determination  of  the  bacterial  content  of 
milk  is  the  estimation  of  the  acidity.  Milk  almost  invariably 
contains  acid-producing  organisms,  and  as  these  find  milk  an 
excellent  medium  for  development  it  would  seem  to  be  logical 
to  assume  that  the  determination  of  the  products  of  bacterial 
metabolism  would  bear  some  relation  to  the  number  of  organisms 
present.  Fred  (vide  supra)  determined  the  acidity  of  200 
samples  of  milk  and  arranged  the  results  into  groups  of  20 
according  to  the  bacterial  count.  His  results  are  given  in 
Table  LIL 

Fred  is  of  the  opinion  that  the  acidity  determination  serves 
a  useful  purpose  in  indicating  to  some  extent  the  proper  dilu- 
tions to  be  used  for  the  bacterial  counts,  and  adds  that  "  the 
relationship  to  the  number  of  bacteria  is  only  approximate." 
Russell  and  Hastings  have  also  suggested  using  this  test  as  a 
guide  to  the  dilutions  to  be  made  in  the  plate  method  and  advise 
10, 100,  and  1,000  dilutions  for  acidities  under  0.2  per  cent  and 
1,000,  10,000  and  100,000  for  acidities  over  0.2  per  cent. 


RELATION  OF  ACIDITY  TO  BACTERIAL  COUNT       133 


TABLE  LII 
RELATION  OF  ACIDITY  TO  BACTERIAL  COUNT  (FRED) 


Group  Number. 

Average  Acidity  as  Lactic 
Acid. 

Number  of  Bacteria  per 
c.cm. 

1 

0.189 

29,647 

2 

0.188 

73,587 

3 

0.183 

160,150 

4 

0.201 

283,250 

5 

0.192 

548,300 

6 

0.205 

1,016,600 

7 

0.206 

1,469,650 

8 

0.212 

2,505,000 

9 

0.231 

4,690,000 

10 

0.250 

8,624,000 

The  author,  during  1914  and  1915,  determined  the  acidity 
and  bacterial  count  of  a  number  of  the  samples  received  for 
routine  examination  with  the  following  results : 

TABLE  LIII 
RELATION  OF  ACIDITY  TO  BACTERIAL  COUNT  (AUTHOR) 


ACIDITY. 

Number  of 

R     t     '  1  C 

Samples. 

Degrees. 

Lactic  Acid, 

48  Hours  at  37°  C. 

Per  Cent. 

34 

14 

0.126 

203,000 

67 

15 

0.135 

332,000 

102 

16 

0.144 

282,000 

144 

17 

0.153 

289,000 

186 

18 

0.162 

232,000 

185 

19 

0.171 

212,000 

120 

20 

0.180 

175,000 

32 

21 

0.189 

408,000 

28 

22 

0.198 

397,000 

9 

23 

0.207 

541,000 

134         THE  ENUMERATION  OF  BACTERIA  IN  MILK 

These  results  show  no  definite  relationship  between  the 
acidity  and  the  bacterial  count  until  the  acidity  approaches 
0.20  per  cent  (22°),  and  in  this  respect,  are  confirmatory  of 
Fred's  results.  Only  9  samples  out  of  a  total  of  917  exceeded 
22°  acidity  and  it  became  obvious  that  the  acidity  determina- 
tion even  as  a  guide  to  the  best  dilutions  to  employ  in  plate 
work  did  not  give  information  commensurate  with  the  labour 
involved.  For  pasteurised  and  heated  milk  the  acidity  estima- 
tion is  of  even  less  value  than  for  ordinary  raw  milk  owing  to 
the  change  in  acidity  acused  by  the  heating  processes. 

BIBLIOGRAPHY 

1.  DelSpine.     Jour,  of  Hyg.     1903,  3,  68. 

2.  Laboratory  Rpt.  of  Chicago  Dept.  of  Health.     1907-1910. 

3.  Race.     Can.  Jour,  of  Pub.  Health.     1915,  6,  13. 

4.  Conn.     Pub.  Health  Rpt.  U.S.A.P.H.S.,     1915,  30,  2390. 

5.  Heinemann  and  Glenn.     Jour.  Inf.  Dis.     1908,  5,  412. 

6.  American  Jour,  of  Pub.  Hyg.     18,  431. 

7.  Clark.     Jour.  Inf.  Dis.     1915,  17,  109-136. 

8.  Slack.     Tech.  Quart.     1906,  19,  No.  1. 

9.  Standard  Methods  for  Bact.  Exam,  of  Milk,  Amer.  Pub.  Health. 

Assoc.,  1912,  p.  25. 

10.  Goodrich.     Jour.  Inf.  Dis.     1914,  14,  512. 

11.  Breed.     Centrabl.  f.  Bakt.,  Abt.  2,  30,  337-340. 

12.  Skar.     Milchw.  Zentbl.     41,  454-461,  ibid.,  705-712. 

13.  Rosam.     Milchw.  Centbl.     1913,  42,  333. 

14.  Conn.     Pub.  Health  Rpt.  U.S.A.P.H.S.     1915,  30,  2394. 

15.  Barthol.     Zeit.  Untersuch.  Nahr.  Genussm.     1908,  15,  385-405. 

16.  Fred.     Zeit.  f.  Bakt.  u.  Parasitenk.     1912,  35,  Abt.  2,  391. 

17.  Fred.     Rpt.  Virginia  Agar.  Expt.  Sta.     1911-12,  206-240. 

18.  Barthol.     Zeit.  Untersuch.  Nahr.  u.  Genussm.     1911,  21,  513-534. 


CHAPTER  VI 
EXCREMENTAL  ORGANISMS 

THE  estimation  of  typical  excremental  organisms  in  milk  is  of 
considerable  value  because  of  the  general  absence  of  these 
bacteria  in  intra-mammary  milk;  they  indicate,  therefore,  the 
amount  of  care  exercised  in  the  production  and  handling  of 
the  milk  in  a  rather  better  manner  than  the  determination  of 
the  total  number  of  organisms,  but  as  milk  drawn  under  the  best 
conditions  is  never  absolutely  free  from  excremental  organisms, 
this  advantage  is  merely  relative. 

The  estimation  of  the  bacteria  usually  regarded  as  indica- 
tive of  manurial  pollution  has  not  in  the  past  been  developed 
to  full  advantage  because  of  the  somewhat  elaborate  technique 
involved,  and  also  because  some  sanitarians  have  regarded  the 
excremental  bacterial  content  as  being  more  determined  by 
duration  and  conditions  of  storage  than  by  the  original  pollu- 
tion. It  would,  undoubtedly,  be  of  great  advantage  if  some 
method  could  be  found  of  determining  the  manurial  pollution 
of  a  sample  at  the  time  of  milking,  not  only  because  it  would 
yield  precise  information  as  to  the  condition  requiring  correc- 
tion, but  also  on  account  of  the  possible  association  of  tubercle 
bacilli  with  the  faecal  bacteria.  Tubercle  bacilli  grow  so  slowly 
in  milk  in  comparison  with  the  typical  excremental  organisms 
that  any  inferential  value  associated  with  the  determination  of 
the  latter  is  rapidly  nullified  by  the  conditions  usually  obtain- 
ing in  the  marketing  of  milk. 

The  organisms  commonly  used  as  indicators  of  manurial 
pollution  are  B.  coli,  B.  enteritidis  sporogenes,  and  Streptococci, 
and  of  these  B.  coli  is  probably  the  most  important  and  the  most 
easily  estimated.  English  bacteriologists  have,  on  the  whole, 

135 


136 


EXCREMENTAL  ORGANISMS 


devoted  more  attention  to  these  estimations  than  their  Ameri- 
can confreres,  but  neither  have  studied  them  as  fully  as  they 
deserve  and  it  is  to  be  hoped  that  this  condition  will  soon  be 
rectified. 

These  organisms  will  now  be  treated  in  detail. 

1.  B.  Coli.  The  term  B.  coli  in  these  pages  is  used  to 
signify  the  general  group  of  aerobic,  non-sporulating  organisms 
that  ferment  lactose  with  the  production  of  acid  and  gas,  and 
not  one  particular  member  of  the  group,  such  as  B.  coli  com- 
munis,  having  certain  specific  characteristics  in  addition  to  the 
generic  ones  just  described.  Many  attempts  have  been  made 
to  regard  certain  members  of  this  group  as  being  more  sig- 
nificant than  others-but  this  has  been  a  comparative  failure 
when  viewed  by  the  light  of  later  experience. 

MacConkey l  reported  upon  the  biochemical  characters  of  a 
number  of  members  of  the  B.  coli  group,  isolated  from  milk  and 
from  the  faeces  of  cows,  and  classified  them  into  four  groups 
according  to  their  action  on  saccharose  and  dulcite.  The 
results  are  given  in  Table  LIV. 

TABLE  LIV 


Milk. 
Per  Cent. 

Cow's  Faeces. 
Per  Cent. 

Saccharose  -}-  du.1  cite  -|-  . 

32  7 

47  9 

Saccharose  —  dulcite  -f-. 

39  2 

25  0 

Saccharose  -(-dulcite  — 

19  6 

12  5 

Saccharose  —  dulcite  —  

8.4 

16  6 

MacConkey  suggested  that  these  groups  should  be  further 
subdivided  according  to  the  ability  to  ferment  adonite  and 
inulin,  the  Voges  and  Proskauer  reaction,  and  the  motility. 
In  1909  he  reported  the  characteristics  of  colon  organisms 
isolated  from  animal  and  human  faeces  and  arranged  the  group- 
ing in  accordance  with  the  subdivision.2  As  this  further 
division  has  not  been  generally  adopted,  the  results  have  been 


B.  COLI 


137 


rearranged  into  the  four  general  groups  in  Table  LV  and  Orr's 
results  3  added  for  comparison. 

TABLE  LV 


MACCONKEY. 

ORR. 

Milk 

Milk 

Human 
Fseces. 

Animal 
Faeces. 

from 
Cow- 

from 
Retailer. 

from 
Con- 

Manure. 

shed. 

sumer. 

Per  Cent. 

Per  Cent. 

Per  Cent. 

Per  Cent. 

Per  Cent. 

Per  Cent. 

Saccharose  +  dulcite  +. 

32.2 

48.1 

28.5 

26.5 

26.1 

18.7 

Saccharose  —  dulcite  +  • 

27.0 

34.3 

13.8 

10.4 

12.8 

35.4 

Saccharose  +dulcite  —  . 

4.5 

43.9 

39.1 

41.1 

33.4 

Saccharose  —dulcite  —  . 

28.0 

8.4 

12.6 

20.4 

16.7 

8.4 

Other  strains  

8.3 

9.2 

1.2 

3.6 

3.3 

4.1 

The  results  of  Rogers  et  al.,4  who  investigated  107  colon 
organisms  obtained  from  milk  products,  and  some  unpublished 
ones  of  the  author  on  the  biochemical  characters  of  coliform 
organisms  obtained  from  226  samples  of  milk,  are  given  in 
Table  LVI. 

TABLE  LVI 


Rogers  et  al. 
Per  Cent. 

Author. 
Per  Cent. 

Saccharose-)-  dulcite  -f-  

24  3 

46  5 

Saccharose  —  dulcite  -j-  . 

14  9 

8  4 

Saccharose  -(-dulcite  — 

37  4 

36  3 

Saccharose  —  dulcite  — 

23  4 

8  8 

The  author's  results,  obtained  with  samples  of  the  Ottawa 
milk  supply,  are  somewhat  in  accordance  with  Orr's  results  as 
regards  the  predominance  of  saccharose  fermenters,  but  show  a 
larger  proportion  of  dulcite  fermenters.  This  predominance  of 
saccharose  fermenters  accords  with  the  results  recorded  for 


138 


EXCREMENTAL  ORGANISMS 


animal  faeces  and  would  seem  to  differentiate  between  animal 
and  human  pollution,  but  as  the  difference  is  one  of  degree  only 
and  is  not  specific,  no  definite  significance  can  be  attached  to  it. 
Although  a  large  amount  of  work  has  been  done  on  the  separa- 
tion of  the  colon  group  of  organisms,  no  test  or  combination  of 
tests  has  been  evolved  that  would  indicate  that  any  one  sub- 
group is  more  typical  than  another,  and  it  must,  therefore,  be 
borne  in  mind  that  to  designate  any  organism  as  being  typical  B. 
coli  because  it  possesses  certain  biochemical  and  morphological 
characteristics  is  a  purely  arbitrary  and  empirical  procedure. 
Moreover,  these  organisms  are  not  to  be  regarded  as  having 
immutable  properties  like  chemical  compounds,  but  to  form 
involution  and  mutation  varieties  according  to  the  environment. 
Milk,  even  when  produced  under  the  best  conditions,  is 
never  quite  free  from  B.  coli,  but  if  reasonable  precautions  are 
taken,  this  group  should  not  be  present  in  25  c.cm.  quantities 
of  byre  milk.  Even  after  bottling  and  delivery  to  the  pur- 
chaser milk  can  be  produced  that  will  average  less  than  two 
B.  coli  per  cubic  centimetre,  even  during  the  summer  months. 
This  is  exemplified  in  Table  LVII. 

TABLE  LVII 
BACTERIA  AND  B.  COLI  IN  CERTIFIED  MILK  (AUTHOR) 


Month. 

Mean  Bacterial 
Count  per  c.cm. 

Mean  B.  Coli 
per  c.cm. 

May  

5,700 

1 

June   ' 

10,900 

2 

July  .  . 

5.000 

0.1 

August                                          .  .  . 

4,500 

0.8 

September 

5,500 

1  4 

When  milk  is  kept  at  a  temperature  not  exceeding  45°  F. 
the  B.  coli  do  not  increase  (vide  p.  104)  and  this  temperature 
may,  therefore,  be  regarded  as  the  critical  anabolic  tempera- 
ture. Above  this  point  they  multiply  rapidly  and  in  summer 


TEMPERATURE  AND     B.  COLI 


139 


the  B.  coli  content  of  milk  must  be  regarded  as  due  more  to 
reproduction  than  to  original  contamination.  Diagram  No. 
Ill,  which  shows  the  B.  coli  content  of  the  Ottawa  raw  milk 
supply  compared  with  the  mean  atmospheric  temperature, 
demonstrates  very  clearly  the  effect  of  temperature.  In  the 
autumn  months  the  curves  do  not  correspond  because  the  mode 
of  the  B.  coli  curve  is  lowered  during  the  hot  summer  months 

DIAGRAM  No.  Ill 

EFFECT  OF  ATMOSPHERIC  TEMPERATURE  ON  B.  COLI  CONTENT 

OTTAWA 


18,000 
16,000 
14,000 

g  12,000 

V 
o 

olO.OOO 

| 

°.  -8,000 

H 

6,000 
4,000 
2,000 

/ 

tf 

^ 

\ 

**CL., 

•-q^ 

/ 

-O 

'1 

\    \ 

*\P 

y 

/ 

Y 

/b 

\ 

/ 

/ 

5 

K 

J 

/ 

B.  c< 
Tern 

ii 

perature  

V 

rC**' 

.'•O 

—  -  o— 

^/ 

Nov.  Dec. 
1914 

Jan.    Feb.   Mar.   Apr.   May  June  July  Aug.  Sept.   Oct. 
1915 

90 

- 1 

70  S 

I 
60  „• 

502 

4 
J 

20 


by  artificial  cooling  of  the  milk  and  the  temperature  of  the  milk 
is,  consequently,  not  proportional  to  the  atmospheric,  but  it  is 
evident  that  artificial  cooling  is  abandoned  before  the  natural 
agencies  become  entirely  operative.  It  is  also  interesting  to 
note  that  after  the  very  cold  winter  weather  the  B.  coli  content 
does  not  increase  until  the  mean  atmospheric  temperature 
exceeds  the  critical  temperature. 


140  EXCREMENTAL  ORGANISMS 

Estimation  of  B.  Coli.  The  methods  in  vogue  for  the  esti- 
mation of  B.  coli  fall  into  two  groups,  (1)  enrichment  methods 
and  (2)  plate  methods. 

Enrichment  Methods.  In  the  enrichment  methods,  varying 
quantities  of  the  sample  are  inoculated  into  liquid  media  and 
incubated,  the  -media  being  subsequently  examined  as  to  the 
presence  or  absence  of  B.  coli.  In  this  test  a  carbohydrate  is 
usually  employed  that  is  fermented  by  B.  coli  with  the  pro- 
duction of  gas  and  special  tubes  are  used  in  which  this  gas  is 
trapped  and  retained  as  visible  evidence  of  fermentation.  On 
account  of  the  economy  of  space  a  small  inverted  tube  con- 
tained in  a  larger  ordinary  culture  tube  (Durham's  tube)  is 
now  in  almost  universal  use  in  the  fermentation  process.  As 
in  water  examination,  there  are  a  number  of  points  in  connec- 
tion with  this  test  that  require  consideration.  The  first  is  the 
composition  of  the  medium  to  be  employed.  If  the  results  are 
to  be  based  on  the  presence  or  absence  of  gas  in  the  tubes,  it 
is  evident  that  lactose  and  not  dextrose  must  be  the  carbo- 
hydrate employed  as  there  are  other  groups  than  B.  coli  that 
ferment  the  latter  sugar.  The  nitrogen  requisite  for  bacterial 
reproduction  is  usually  supplied  by  the  addition  of  peptone, 
although  this  may  be  partially  displaced  by  sugar-free  beef 
infusion  or  extract.  Potassium  chloride  is  also  a  desirable  con- 
stituent (Chamot  and  Sherwood).  Such  a  medium  will  give 
gas  formation  even  with  attenuated  B.  coli,  and,  if  only  vigorous 
forms  are  desired  to  be  estimated  the  medium  can  be  prepared 
with  a  base  of  fresh  ox  bile  instead  of  water.  There  is  con- 
siderable evidence,  however,  that  the  lactose  ox-bile  medium 
inhibits  the  growth  of  a  number  of  vigorous  forms  of  B.  coli  in 
addition  to  the  attenuated  ones  and  for  this  reason  the  fresh  bile 
medium  is  often  regarded  with  disfavour.  MacConkey's  me- 
dium, containing  0.5  per  cent  of  bile  salt,  may  also  be  used  and 
in  this  case  the  results  will  usually  be  intermediate  between 
those  obtained  with  lactose  broth  and  lactose  bile.  The  main 
objection  to  lactose  broth  is  the  excessive  number  of  anomalies 
caused  by  the  overgrowth  of  other  organisms.  Aciduric  bacilli 


ESTIMATION  OF  B.  COLI  141 

occasionally  reproduce  so  rapidly  in  the  lower  dilutions  as  to 
prevent  the  growth  of  the  coliform  bacteria  and  so  give  a 
negative  gas  test  when  a  much  higher  dilution  of  the  same 
sample  shows  copious  gas  formation. 

The  usual  amounts  of  lactose  and  peptone  employed  in  the 
fermentation  test  are  1  per  cent  of  each,  but  Chamot  and  Sher- 
wood 5  have  shown  that  a  lactose  content  of  0.6  per  cent  pro- 
duces equally  satisfactory  results  as  1.0  per  cent.  Under  0.6 
per  cent  the  results  were  irregular  and  the  total  volume  of  gas 
small,  whilst  quantities  much  exceeding  1.0  per  cent  retarded 
the  rate  of  gas  formation.  With  normal  acidities  they  found 
that  the  total  gas  volume  was  proportional  to  the  concentration 
of  the  nitrogen  whether  present  as  peptone,  beef  extract  or 
infusion.  With  increasing  amounts  of  peptone  the  increase 
in  gas  volume  was  rapid  until  4.0  per  cent  was  reached  and  when 
both  final  volume  and  rate  of  production  were  considered, 
it  was  found  that  a  concentration  of  3.0  to  4.0  per  cent  was  the 
optimum.  Potassium  chloride  (0.6  per  cent)  hastened  gas 
formation  and  was  found  superior  to  phosphates  and  other 
salts.  The  concentrations  finally  recommended  were  lactose 
0.8  per  cent,  peptone  3  to  4  per  cent,  KC1  0.6  per  cent,  and  the 
reaction  +1.0  per  cent.  With  lactose  bile  the  nitrogen  content 
should  be  sufficient  with  the  addition  of  only  1.0  per  cent  of 
peptone,  but  in  other  media  the  higher  amount  should  be  em- 
ployed. For  the  concentration  method  the  author  uses  ordinary 
lactose  broth  or  lactose  bile  salt  broth  in  preference  to  lactose 
bile  on  account  of  the  irregularities  often  found  with  lactose 
bile  and  due  to  the  variations  in  composition. 

The  number  of  tubes  to  be  employed  in  order  to  obtain 
reasonably  precise  results  is  the  second  point  for  consideration. 
It  has  been  usual  to  use  such  dilutions  of  milk  that  the  quan- 
tities represent  decimal  fractions  of  1  c.cm.  and  to  endeavour 
to  obtain  at  least  one  positive  and  one  negative  result.  Al- 
though, in  many  instances,  no  attempt  has  been  made  to  con- 
vert such  positive  and  negative  findings  into  mathematical 
expressions,  others  have  attempted  to  do  so  by  taking  the 


142  EXCREMENTAL  ORGANISMS 

reciprocal  of  the  lowest  quantity  showing  a  positive  result  as 
representing  the  number  of  B.  coli  per  cubic  centimetre.  Thus, 
0.1  c.cm.+,  0.01  c.cm+,  0.001  c.cm.  — ,  was  expressed  as  100  B. 
coli  per  cubic  centimetre.  When  the  average  of  a  number  of 
samples  from  one  source  is  calculated  by  this  method  (Phelps  6) 
an  accurate  result  is  obtained  providing  the  series  is  fairly 
large  (about  25),  but  McCrady  7  has  shown  that  for  individual 
samples  such  assumptions  are  far  from  accurate.  McCrady 
calculates  from  the  theory  of  probabilities  that  the  most  prob- 
able number  of  B.  coli  present  per  cubic  centimetre,  if  the  above 
result  were  obtained,  would  be  230  and  not  100  as  assumed.  It 
is  possible  that  any  number  of  B.  coli  per  cubic  centimetre  would 
produce  this  result  and,  in  order  to  reduce  the  range  of  possibili- 
ties and  sharpen  the  probability  curve,  it  becomes  necessary  to 
employ  more  than  one  tube  of  each  dilution.  The  greater  the 
number  of  tubes  used  the  greater  is  the  precision  obtained.  With 
a  milk  of  unknown  origin  that  may  contain  up  to  100,000  B.  coli 
per  cubic  centimetre  it  is  obvious  that  even  if  only  three  tubes  of 
each  dilution  are  used  the  total  number  of  tubes  for  each  sample 
becomes  so  great  as  to  be  cumbersome.  For  this  reason  the  tube 
method  of  estimating  B.  coli  in  milk  cannot  be  recommended. 
The  third  point  for  consideration  is  the  method  of  recording 
the  results.  If  desired,  all  tubes  showing  gas  may  be  plated  out 
on  rebipelagar  or  litmus  lactose  agar  and  the  red  colonies  so 
obtained  put  through  confirmatory  tests,  but  as  such  a  pro- 
cedure requires  much  time  and  labour  it  will  be  found  more 
convenient  and  fairly  accurate  to  record  all  tubes  as  positive 
that  show  more  than  5  per  cent  of  gas.  Anomalies  at  the 
higher  end  of  the  series  should  be  ignored  as  they  are  probably 
the  result  of  overgrowths,  but  those  at  the  lower  end  should 
be  corrected  by  moving  the  lower  positive  results  to  the  next 
higher  dilution;  thus,  1.0  c.cm.  —  ,  0.1  c.cm.+,  0.01  c.cm.+, 
0.001  c.cm.+,  should  be  recorded  as  1.0  c.cm.+,  0.1  c.cm.-f, 
0.01  c.cm. + ,  0.001  c.cm.-f,  but  1.0  c.cm.+,  0.1  c.cm.+, 
0.01  c.cm.  —  ,  0.001  c.cm.+,  should  be  recorded  as  1.0  c.cm.-|-, 
0.1  c.cm.+,  0.01  c.cm.+,  0.001  c.cm.-. 


ESTIMATION  OF  B.  COLI  143 

Plate  Methods.  Quite  a  number  of  solid  media  have  been 
suggested  for  the  isolation  and  enumeration  of  B.  coli  and  allied 
organisms  and  of  these  the  most  useful  are  Endo's  medium 
(fuchsin  sulphite  agar),  Drigalski  and  Conradi's  medium  (nut- 
rose  agar),  sesculin  bile  salt  agar,  and  rebipelagar  (neutral 
red  bile  salt  agar).  On  account  of  the  difficulties  connected 
with  the  preparation  and  use  of  the  first  two  media  the 
author  prefers  the  latter  two.  These  are  easy  to  prepare  (see 
appendix  p.  207)  and  may  be  used  in  exactly  the  same  manner 
as  ordinary  nutrient  agar  or  gelatine.  The  Committee  on 
Standard  Methods  of  Milk  Analysis  of  the  American  Public 
Health  Association  investigated  the  latter  two  media  and 
reported  in  favour  of  the  sesculin  medium.  They  found  more 
bacteria  of  the  B.  coli  group  on  rebipelagar  in  nearly  every 
instance  but  this  was  due  to  the  difficulty  in  deciding  which 
were  the  coliform  colonies  on  the  sesculin  medium.  Of  more 
than  fifty  colonies  subcultured  from  the  neutral  red  medium 
only  67  per  cent  were  found  to  be  B.  coli  or  B.  serogenes  (B. 
lactis  serogenes)  whereas  all  the  dark  colonies  from  the  sesculin 
medium  were  of  the  B.  coli  family.  Savage  9,  from  his  expe- 
rience with  sesculin  agar  and  rebipelagar,  as  compared  with 
lactose  bile  salt  broth,  has  expressed  the  opinion  that  both 
media  are  equally  useful  but  inferior  to  L.  B.  B.  tubes  on 
account  of  the  difficulty  in  arriving  at  accurate  estimations 
of  the  numbers  by  direct  plating.  The  author  has  had  very 
little  experience  with  sesculin  agar,  but  the  extended  observa- 
tions that  he  has  made  with  rebipelagar  do  not  entirely  agree 
with  the  above  results.  A  series  of  comparative  experiments 
on  100  samples  with  rebipelagar  and  lactose  bile  salt  broth 
gave  the  following  results,  gas  formation  being  regarded  as 
evidence  of  the  presence  of  B.  coli  in  the  tube  series  without 
confirmation. 

Medium.  B.  coli  per  C.cm. 

Rebipelagar 15,326 

Lactose  broth 10,182 


144  EXCREMENTAL  ORGANISMS 

In  72  samples  the  two  methods  agreed,  that  is  the  plate 
count  was  in  approximate  agreement  with  the  reciprocal  of  the 
smallest  quantity  of  the  sample  showing  gas  formation.  In  25 
samples  the  results  differed  by  one  dilution  (the  dilutions  being 
decimal  fractions  of  a  cubic  centimetre),  in  two  samples  by  two 
dilutions,  and  in  one  sample  by  three  dilutions.  The  agree- 
ment in  the  averages  is  very  reasonable  when  the  chance  errors 
of  distribution  inherent  to  the  tube  method  are  considered,  and 
the  differences  between  individual  samples  can  be  shown  to  be 
well  within  the  limits  calculated  by  the  theory  of  probabilities. 

The  errors  connected  with  rebipelagar  are  caused  (1)  by  the 
destruction  of  the  characteristic  colour  of  the  B.  coli  colonies  by 
the  diffusion  of  amines  or  other  alkaline  substances  through  the 
medium  and  (2)  by  the  development  of  red  colonies  by  organ- 
isms not  of  the  B.  coli  group.  When  a  dilution  of  the  sample 
is  employed  that  prevents  overcrowding  of  the  colonies,  the 
first  error  is  usually  avoided  unless  there  is  a  large  excess  of 
alkali  forming  organisms  present;  this  condition  can  be  easily 
recognised  because  either  a  yellow  area  is  produced  concen- 
trically from  a  colony,  or,  as  is  usually  the  case,  the  whole-  of 
the  medium  is  yellow.  The  error  due  to  organisms  other  than 
coliform  bacteria  is  small  and  can  be  largely  eliminated  by 
experience.  The  characteristic  forms  produced  by  coliform 
organisms  on  the  surface  of 'the  plate  may  either  be  a  colony 
of  deep  red  colour  producing  a  haze  in  the  surrounding  medium, 
or  one  with  a  red  centre  surrounded  by  a  yellowish  or  pinkish 
aureole  of  slimy  consistency.  The  subsurface  colonies  are  of 
the  former  variety  but  may  not  invariably  produce  the  haze 
which  is  due  to  the  diffusion  of  acid  into  the  surrounding 
medium.  The  author,  during  the  examination  of  several 
hundreds  of  coliform  colonies  from  milk  plated  on  rebipelagar, 
has  only  met  with  two  organisms,  one  a  coccus  and  the  other  a 
bacillus,  that  produced  colonies  resembling  those  typical  of 
B.  coli,  but  many  organisms  that  ferment  lactose  with  the  pro- 
duction of  acid  may,  especially  after  prolonged  incubation, 
produce  colonies  that  bear  a  superficial  resemblance  to  those 


CLASSIFICATION  OF  B.  COLI  TYPE  145 

described  above.  There  is  also  a  danger  of  mistaking  pin  point 
red  colonies  produced  by  acid-forming  streptococci  for  those 
produced  by  attenuated  B.  coli  and  it  will  be  found  advisable 
to  ignore  all  such  colonies  when  examining  the  plates.  By 
this  procedure,  only  organisms  in  a  fairly  vigorous  state  are 
counted,  and,  although  it  is  somewhat  empirical  in  character, 
it  produces  results  that  are  of  greater  sanitary  significance. 
Of  271  red  colonies  fished  from  rebipelagar,  the  author  found 
that  236  (87  per  cent)  were  of  the  B.  coli  group  so  that  even  if 
all  the  red  colonies  are  counted  no  serious  errors  will  be  intro- 
duced. 

One  difficulty  in  connection  with  the  use  of  rebipelagar  is  the 
quality  of  the  bile  salt.  Many  brands  of  this  salt  are  pur- 
chasable but  very  few  are  satisfactory.  Sodium  taurocholate, 
sodium  glycocholate,  and  many  brands  of  the  commercial  bile 
salt  are  too  restrictive  in  their  action  on  B.  coli  and  if  the 
amount  is  reduced  to  avoid  this,  the  selective  action  is  de- 
stroyed. With  bile  salt  of  satisfactory  quality,  vigorous  B. 
coli  will  produce  colonies  3  to  5  mm.  in  diameter  in  twenty- 
four  hours  at  37°  C.  and  all  brands  that  fail  to  do  this  should 
be  rejected. 

Classification  of  B.  Coli  Type.  It  has  been  indicated  earlier 
in  this  chapter  (page  136)  that  an  attempt  to  regard  one  par- 
ticular type  of  B.  coli  as  having  more  sanitary  significance 
than  others  has  been  a  comparative  failure.  The  present 
problem  is  not  the  definition  of  the  properties  of  a  distinct 
biotype  such  as  B.  coli  communis  or  B.  acidi  lactici  but  the 
correlation  of  properties  with  the  immediate  previous  environ- 
ment. The  faecal  types  of  B.  coli  can  apparently  be  distin- 
guished from  those  occurring  on  grain  n  by  the  hydrogen  ion 
concentration  produced  in  dextrose  broth  containing  0.5  per 
cent  of  dextrose,  1.0  per  cent  of  peptone,  and  0.2  per  cent  of  acid 
potassium  phosphate.  This  can  best  be  determined  by  the 
methyl  red  reaction  of  Clark  and  Lubs 12  which  Levine  13  has 
shown  to  be  correlated  with  the  Voges  and  Proskauer  reaction. 
The  precise  sanitary  significance  of  these  so-called  grain  types 


146  EXCREMENTAL  ORGANISMS 

has  yet  to  be  determined  but  the  present  trend  of  opinion  is 
towards  the  view  that  the  methyl  red  negative,  Voges  and 
Proskauer  positive  types  (grain  types)  are  harmless  sapro- 
phytes. The  members  of  the  B.  coli  group  derived  from  human 
and  bovine  hosts  can  be  partially  distinguished  by  the  usual 
reactions  in  sugar  broths,  the  proteoclastic  cleavage  of  gelatine, 
and  the  production  of  indol  from  peptone,  but  these  reactions 
are  not  sufficiently  specific  for  routine  work  although  they  have 
a  limited  application  for  research  purposes. 

2.  B.  Enteritidis  Sporogenes.  As  the  spores  of  B.  enteri- 
tidis  sporogenes  are  present  in  considerable  quantities  in 
manure  and  do  not  multiply  in  milk,  the  estimation  of  these 
would  constitute  an  admirable  test  for  original  pollution  if  all 
other  sources  of  these  spores  could  be  eliminated.  The  spores, 
however,  may  be  derived  from  dirty  vessels  and  in  practice  it 
is  found  that  milk  cans  form  a  most  fruitful  source  of  these 
organisms.  Milk  cans,  unless  thoroughly  sterilised  with  live 
steam,  are  very  liable  to  contain  large  numbers  of  spores  of 
various  organisms  as  the  treatment  given,  though  usually 
sufficiently  severe  to  kill  the  non-sporulating  organisms,  is  not 
drastic  enough  to  kill  the  spores.  The  usual  temperature  at 
which  milk  is  pasteurised  (143°-145°  F.)  is  also  not  sufficiently 
high  to  kill  the  spores,  so  that  the  spore  test  is  of  considerable 
value  in  arriving  at  an  opinion  as  to  the  bacteriological  condi- 
tion of  pasteurised  milk  previous  to  pasteurisation.  This  test 
is,  however,  of  much  smaller  value  than  the  direct  microscopical 
test  previously  described. 

For  the  estimation  of  B.  enteritidis  sporogenes  spores, 
various  quantities  of  the  milk  are  measured  out  into  sterile 
test  tubes,  heated  in  a  water  bath  at  80°  C.  for  fifteen  minutes, 
cooled,  and  incubated  anserobically  at  37°  C.  To  obtain 
anaerobic  conditions  the  tubes  may  be  placed  in  an  air-tight  jar 
containing  alkaline  pyrogallic,  but  satisfactory  results  may 
be  obtained  by  covering  the  surface  of  the  sample  in  each  tube 
with  paraffine;  it  is  rather  doubtful  whether  even  this  precau- 
tion is  necessary,  as  the  butter  fat  which  rapidly  rises  and  seals 


STREPTOCOCCI 


147 


the  surface  usually  produces  the  necessary  conditions.  The 
method  of  Savage  10  is  the  most  suitable  with  regard  to  the 
quantities  of  the  sample  to  be  tested.  He  suggests  using  ten 
tubes  and  placing  2  c.cms.  in  each  tube,  but  this  quantity  may 
of  course  be  varied  in  accordance  with  the  nature  of  the  sample. 
It  is  decidedly  preferable  to  use  a  number  of  tubes  containing 
small  amounts  of  milk  than  only  a  few  tubes  containing  larger 
amounts  (vide  supra).  After  two  days  incubation  the  tubes  are 
examined  for  the  "  enteritidis  change  "  which  is  indicated  by  a 
complete  separation  of  the  curd  and  the  production  of  acid, 
the  latter  being  easily  detected  by  litmus  solution.  As  other 
organisms,  such  as  B.  butyricus,  give  this  reaction,  it  is  not  to  be 
entirely  relied  upon,  but  these  organisms  are  mainly  non- 
pathogenic  and  may  be  differentiated  by  injecting  1  c.cm.  of 
the  whey  subcutaneously  into  a  guinea  pig. 

Using  ten  tubes  containing  2  c.cms.  each,  the  most  probable 
number  of  spores  present  in  100  c.cms.  of  sample  for  each  pos- 
sible result  is  given  in  the  Table  LVIII,  which  is  adapted  from 
McCrady's  results.7 

TABLE  LVIII 


Result.     Positive  Tubes. 

Most  Probable  Number  of 
Spores  per  100  c.cms. 

A 

0 

A 

5 

A 

11 

A 

17 

A 

25 

A 

34 

A 

45 

A 

60 

A 

80 

A 

114 

« 

Over  114 

3.  Streptococci.     Cow  manure  contains  100,000  to  10,000-, 
000,000  streptococci  per   gram,  and  the   estimation  of  these 


148  EXCREMENTAL  ORGANISMS 

organisms  in  milk  was  long  ago  suggested  as  a  means  of  deter- 
mining manurial  pollution,  but,  after  considerable  work  had 
been  done  on  the  nature  and  significance  of  the  streptococci 
usually  found  in  milk  this  test  fell  into  general  desuetude.  It 
was  found  that  milk  drawn  under  the  best  aseptic  conditions 
contained  streptococci  which  found  milk  an  excellent  nidus  for 
reproduction  and  that  it  was  practically  impossible  by  simple 
tests  to  distinguish  these  organisms  from  those  derived  from 
manure.  The  examination  of  milk  for  Str.  lacticus  and  Str. 
pyogenes  will  be  discussed  later,  but  it  may  be  stated  here  that 
the  identification  of  these  organisms  is  far  from  being  reliable 
and  that  their  significance  is  still  an  open  question. 

For  the  estimation  of  streptococci,  varying  dilutions,  as  in 
the  enrichment  method  for  B.  coli,  are  inoculated  into  neutral 
red  dextrose  broth  tubes  and  incubated  at  37°  C.  for  two  days. 
The  sediment  is  then  examined  microscopically  for  long  chains 
by  means  of  a  .hanging  drop  preparation  and  all  doubtful  cases 
confirmed  by  stained  smears.  If  desired,  the  streptococci  may 
be  isolated  in  pure  culture,  and  the  morphological  and  bio- 
chemical characteristics  determined  by  spreading  the  diluted 
sediment  over  ordinary  nutrient  agar  or  whey  agar  and  fishing 
off  the  isolated  colonies  after  incubation.  The  properties  of 
Str.  bovis,  Str.  equinus  and  Str.  fsecalis  are  given  in  Table  LIX 
on  page  155.  The  criticism  made  above  with  regard  to  the 
tube  method  for  expressing  a  numeral  value  for  B.  coli  applies 
equally  to  this  method  for  estimating  streptococci.  As  prob- 
ably only  excessive  numbers  of  fsecal  streptococci  have  any 
sanitary  significance,  the  examination  of  a  direct  smear  as  in 
the  Breed  method  for  estimating  the  total  number  of  bacteria 
or  of  a  smear  from  a  centrifugalised  deposit,  will  give  equally 
good  results  with  less  expenditure  of  time  and  labour. 


BIBLIOGRAPHY  149 


BIBLIOGRAPHY 

1.  McConkey.     Jour,  of  Hyg.     1906,  6,  385. 

2.  McConkey.     Jour,  of  Hyg.     1909,  9,  86. 

3.  Orr.     Rpt.  on  an  investigation  as  to  the  contamination  of  milk. 

London,  1908. 

4.  Rogers  et  al.     J.  Inf.  Dis.     1914,  14,  411-475. 

5.  Chamot  and  Sherwood.     J.  Amer.  Chem.  Soc.     1915,  37,  1949-59. 

6.  Phelps.     Amer.  Pub.  Health  Assoc.  Rpt.     33,  9. 

7.  McCrady.     J.  Inf.  Dis.     1915,  17,  183-212. 

8.  Rpt.  of  Amer.  Pub.  Health  Assoc.,  Amer.  J.  of  Pub.  Health.     18,  431. 

9.  Savage.     Milk  and  the  Public  Health.     London,  1914,  10,  163. 

10.  Savage.     Ibid.,  p.  189. 

11.  Rogers  et  al.     Jour.  Inf.  Dis.     1915,  17,  137. 

12.  Clark  and  Lubs.     Jour.  Inf.  Dis.     1915,  17,  160. 

13.  Levine.    Jour.  Inf.  Dis.    1916,  18,  358. 


CHAPTER  VII 
PATHOGENIC  ORGANISMS 

Streptococci.  Although  the  etiological  relation  of  septic 
sore  throat  to  infected  milk  has  been  noted  on  many  occasions 
in  Great  Britain  during  the  past  thirty  years,  it  is  only  during 
the  past  decade  that  any  systematic  investigations  have  been 
carried  out  and  the  bacteriology  of  this  pathological  condition 
developed.  Probably  the  first  bacteriological  examination  of 
any  note  was  made  in  connection  with  the  Angelsey  outbreak 
of  1897  1  when  it  was  reported  that  Staphylococcus  pyogenes 
and  Streptococcus  pyogenes  were  found  in  the  milk  but  no  B. 
diphtherias.  Examination  of  the  patients'  throats  gave  similar 
results.  Some  of  the  most  important  contributions  to  the 
bacteriology  of  septic  sore  throat  are  those  of  Savage.2  Of  the 
36  cases  of  mastitis  investigated,  21,  or  68  per  cent  were  due  to 
streptococci,  5,  or  16  per  cent  to  staphylococci,  and  the  re- 
mainder to  B.  coli,  B.  tuberculosis  and  unclassified  causes. 
On  cultivation  of  the  streptococci  in  the  usual  Gordon  test 
media,  it  was  found  that  a  large  percentage  was  of  one  type, 
called  by  Savage,  Streptococcus  mastiditis.  This  type  tended 
to  long  chain  formation  and  grew  luxuriantly  in  broth  forming  a 
flocculent  deposit  above  which  the  supernatant  liquid  remained 
clear.  Lactose,  dextrose,  and  saccharose  were  invariably  fer- 
mented with  the  production  of  acid,  and  occasionally  salacin, 
raffinose,  and  inulin.  Mannite  was  never  fermented.  In  milk 
acid  was  produced  and  a  clot  formed  within  three  days ;  gelatin 
was  not  liquefied  and  no  neutral  red  reaction  was  produced. 
It  was  non-pathogenic  to  mice.  In  16  cases  of  sore  throat 
Savage  found  the  two  chief  varieties  of  streptococci  to  corre- 
spond to  Andrewes  and  Holder's  Str.  anginosus  and  Str.  pyo- 

150 


STREPTOCOCCI  151 

genes  types  with  the  former  predominating  (vide  p.  155). 
The  bovine  type  Str.  mastiditis,  and  the  human  type  Str. 
anginosus  he  was  unable  to  distinguish  either  morphologically 
or  biochemically,  but  a  marked  difference  in  virulence  was 
found  on  animal  injection.  By  auto  inoculation  on  the  tonsils 
Savage  was  unable  to  produce  either  local  or  general  symptoms 
with  Str.  mastiditis  even  when  massive  doses  were  employed, 
and,  in  general,  the  organisms  could  only  be  recovered  with 
difficulty  even  after  such  a  short  period  as  two  to  three  days. 
The  author  has  been  unable  to  find  any  record  of  any  tests  being 
made  by  Savage  as  to  the  hsemolytic  properties  of  the  organisms 
isolated  by  him;  this  is  of  considerable  importance,  as  haemolysis 
is  now  generally  regarded  as  characteristic  of  the  pathogenic 
types  Str.  pyogenes  and  Str.  anginosus. 

Until  1911  septic  sore  throat  seems  to  have  been  passed 
unrecognised  in  America,  but  the  Boston  epidemic  in  that  year, 
with  over  2000  cases,  gave  an  impetus  to  the  study  of  this  disease, 
and  since  then  it  has  proved  to  be  one  of  the  most  fertile  fields 
for  research  work.  In  the  Boston  epidemic,  as  in  the  later  ones 
at  Chicago,  Baltimore,  Concord  (N.  H.)  and  other  places,  the 
origin  was  traced  to  the  milk  supply  and  it  was  circumstantially 
established  that  the  specific  cause  was  a  hsemolytic  strepto- 
coccus of  the  pyogenes  variety. 

Krumwiede  and  Valentine3  investigated  an  outbreak  of 
septic  sore  throat  on  Long  Island  in  1914  and  reported  that  it 
was  caused  by  the  transfer  of  pathogenic  streptococci  from  a 
case  of  sore  throat  on  a  farm  to  one  of  the  cows  in  the  herd.  An 
examination  of  the  herd  showed  that  five  cows  were  giving  milk 
containing  a  moderate  number  of  streptococci  from  one  or  more 
quarters  and  that  one  of  these  gave  physical  evidence  of  mas- 
titis. All  these  streptococci,  however,  were  non-hsemolytic, 
but  one  other  cow  was  found  in  which  were  moderate  numbers 
of  haemolytic  streptococci  in  two  quarters  and  enormous  num- 
bers in  a  third  quarter.  The  milk  from  this  quarter  was  floc- 
culent.  These  streptococci  were  morphologically  and  bio- 
chemically identical  with  those  isolated  from  the  throats  of  the 


152  PATHOGENIC  ORGANISMS 

sufferers  in  the  epidemic  and  from  the  probable  original  case. 
These  organisms  were  of  the  Str.  pyogenes  type  and  fermented 
salicin  but  not  raffinose  or  mannite. 

Another  link  in  the  chain  of  evidence  in  favour  of  the 
streptococcal  origin  of  these  outbreaks,  was  founded  by  Jack- 
son,4 who  showed  that  experimental  arthritis  could  be  pro- 
duced in  rabbits  by  the  intravenous  injection  of  hsemolytic 
streptococci.  This  is  important  on  account  of  the  frequency  of 
joint  infection  as  a  sequel  to  septic  sore  throat  as  noted  by 
many  observers  in  the  various  epidemics. 

Davis  and  Capps  5  endeavoured  to  produce  an  experimental 
infection  of  milk  by  smearing  the  uninjured  teats  of  a  cow  with 
typical  haemolytic  streptococci  recently  isolated  from  a  ease  of 
streptococcal  tonsilitis;  this  was  unsuccessful,  but  on  repeating 
the  experiment  after  previously  abrading  the  end  of  the  teat 
near  the  meatus,  an  infection  occurred  and  streptococci  and 
leucocytes  were  found  in  abundance  in  the  milk  of  the  infected 
quarter.  Similar  results  were  produced  by  injecting  the  cul- 
ture into  the  udder. 

In  view  of  the  strong  evidence  that  milk-borne  streptococci 
were  causative  agents  of  septic  sore  throat  it  became  imperative 
that  a  study  should  be  made  of  the  streptococci  which  are 
invariably  found  in  milk,  even  though  produced  under  the  best 
conditions,  in  order  to  ascertain  if  there  were  any  relation  be- 
tween these  facts.  Heinemann6  has  shown  that  Str.  lacticus 
occurs  constantly  in  milk  and  that  the  morphological  and  bio- 
chemical characteristics  of  this  organism  on  ordinary  media 
are  identical  with  those  of  Str.  pyogenes.  Later  7  he  found 
that  by  repeated  passage  through  rabbits,  he  was  able  to  exalt 
the  virulence  of  Str.  lacticus  to  such  an  extent  that  compara- 
tively small  doses  were  fatal.  The  lesions  produced  were  very 
similar  to  those  produced  in  human  beings  by  Str.  pyogenes. 
Miiller  8  found  that  milk  streptococci  and  pathogenic  strep- 
tococci showed  no  material  difference  in  their  agglutination  and 
hsemolytic  properties  but  differed  widely  in  the  rapidity  with 
which  they  coagulated  milk.  Heinemann  in  1915  9  reported 


EXAMINATION  FOR  STREPTOCOCCI  153 

the  results  of  further  experiments  on  the  pathogenicity  of  Str. 
lacticus  and  these  in  general  confirm  his  earlier  work.  Two 
strains,  one  only  of  which  was  hsemolytic,  but  both  capable  of 
fermenting  a  variety  of  the  usual  test  substances,  were  exalted 
in  virulence  by  animal  passage,  and  it  is  important  to  note  that 
the  fermentative  capacity  gradually  decreased  until  finally  one 
strain  fermented  only  dextrose,  and  the  other  dextrose  and 
saccharose.  The  non-hsemolytic  strain  became  hsemolytic  and 
both  showed  an  increased  tendency  to  chain  formation.  From 
these  results  Heinemann  suggests  that  the  determination  of  the 
fermentative  ability  of  the  streptococci  might  be  of  value  in 
determining  the  previous  environment  of  the  organisms.  If 
in  contact  with  an  animal  lesion  a  low  fermentative  capacity 
would  result  whilst  a  high  capacity  would  indicate  a  medium 
rich  in  carbohydrates. 

Although  the  questions  of  the  variability  of  streptococci 
in  mastitis  and  the  relation  of  mastitis  to  septic  sore  throat, 
are  still  far  from  being  satisfactorily  solved,  it  has  been  fairly 
definitely  established  that  the  great  majority  of  the  strep- 
tococci ordinarily  found  in  milk  are  non-pathogenic  and  do 
not  indicate  a  pathological  condition  of  the  udder.  Str.  lac- 
ticus, which  may  be  found  in  almost  every  sample  of  milk,  is 
used  industrially  in  cheese  manufacture  and  is  also  employed 
as  a  therapeutic  agent.  This  streptococcus  is  typical  of  the 
group  characterised  by  high  fermentative  capacity  and  low 
pathogenicity.  The  pathogenic  streptococci,  on  the  other 
hand,  ferment  but  few  of  the  Gordon  test  substances  and  pro- 
duce low  acidities  in  the  media  that  are  fermented;  the  mor- 
phological appearance  is  characterised  by  the  picket  fence 
(stalkett)  formation  but  the  chain  may  be  either  short  or  long; 
haemolysis  is  marked. 

Examination  for  Streptococci.  Probably  the  most  satis- 
factory method  of  examination  for  excessive  numbers  of  strep- 
tococci resulting  from  mastitis,  "is  the  direct  miscroscopical 
method  of  a  smear  prepared  either  by  the  Stewart-Sloan  method 
described  on  page  126  or  the  Breed  method  described  on  page 


154  PATHOGENIC  ORGANISMS 

129.  In  the  microscopical  examination,  the  streptococci  having 
the  typical  form  of  Str.  lacticus  (elongated  cocci,  usually  in 
pairs)  should  be  ignored  and  a  search  made  for  the  picket  fence 
variety  only.  These,  on  staining  with  methylene  blue,  usually 
appear  in  chains  with  solidly  stained  portions  at  right  angles 
to  the  longitudinal  axis;  capsules  are  usual  but  are  not  invari- 
ably found.  Some  observers  attach  more  significance  to  the 
long-chain  types,  but  in  view  of  the  numerous  cases  in  which  the 
short-chain  types  have  been  associated  with  pathological  con- 
ditions, it  would  appear  to  be  good  policy  to  attach  equal 
significance  to  both  varieties.  The  property  of  chain  forma- 
tion is  undoubtedly  a  variable  one  and  is  profoundly  modified 
by  the  composition  of  the  medium  and  general  environment. 

In  the  indirect  method,  the  sample  is  diluted  as  in  the  exam- 
ination for  faecal  streptococci  and  the  various  dilutions  seeded 
into  dextrose  broth.  After  incubation  for  forty-eight  hours 
at  37°  C.,  the  cultures  are  examined  for  chain  formation  by 
making  a  smear  or  a  hanging  drop  preparation;  from  the 
smallest  quantity  containing  typical  chains  the  approximate 
number  of  streptococci  can  be  calculated.  If  desired,  the  broth 
cultures  can  be  plated  out  on  nutrient  agar  or  gelatine,  and  the 
organisms  isolated  in  pure  culture.  The  quickest  and  most 
satisfactory  method  of  examination  for  pathogenic  streptococci 
is  by  plating  on  blood  agar.  Ruediger41  as  early  as  1912 
suggested  the  differentiation  of  Str.  pyogenes  from  Str.  lacticus 
by  the  haemolytic  properties  of  the  former  and  since  that  date 
several  workers  have  demonstrated  that  haemolysis  is  a  usual 
property  of  the  pathogenic  streptococci.  All  haemolytic  strains, 
however,  are  not  pathogenic. 

The  best  technique  is  to  add  various  dilutions  of  the  sample 
to  10  c.cms.  of  meat  infusion  agar  containing  1  c.cm.  of  horse 
blood  and  then  pour  into  Petri  plates.  These  are  incubated 
at  37°  C.  and  examined  after  twenty-four  and  forty-eight  hours 
for  haemolysis.  Those  colonies  showing  a  clear,  transparent, 
colourless  zone  are  transferred  to  broth  and  finally  inoculated  in 
the  usual  Gordon  test  media,  viz.,  dextrose,  saccharose,  raf- 


EXAMINATION  FOR  STREPTOCOCCI 


155 


finose,  mannite,  lactose,  and  salicin  broths  for  determination  of 
acidity,  in  milk  for  coagulation,  and  to  blood  agar  plates  for 
haemolysis.  A  virulence  test  is  also  desirable,  but  in  considering 
the  results  obtained  due  regard  must  be  given  to  the  dosage  and 
method  of  inoculation.  A  quantity  of  broth  that  is  sufficient  to 
kill  the  test  animal  in  three  days  when  injected  intravenously 
might  not  produce  more  than  local  symptoms  when  given  sub- 
cutaneously,  and  similar  conditions  apply  to  the  dosage.  For 
guinea  pigs  1  c.cm.  of  a  forty-eight  hour  broth  culture  and  for 
mice  0.5  c.cm.  of  a  twenty-four  hour  culture  have  been  found 
to  give  satisfactory  results  when  injected  into  the  peritoneal 
cavity. 

The  biochemical  characteristics  should  be  determined 
quantitatively  by  Winslow's  method  39  if  the  best  results  are  to 
be  secured. 

TABLE  LIX 

BIOCHEMICAL  CHARACTERS  OF  PRINCIPAL  TYPES  OF 
STREPTOCOCCI.     (BROADHURST) 


a' 

o 

Name  of  Variety. 

I 

, 

§ 

oJ 

0 

.3 

1 

If 

Type  Named  by 

X 

0 

2 

1 

a 

'3 

a' 

'o 

*3 

a 

II 

Q 

• 

£ 

1 

3 

1 

w 

0 

Str.  equinus.  .  . 

X 

o 

X 

o 

o 

X 

— 

— 

Andrews  and 

Str.  niitis  

x 

x 

x 

o 

o 

x 





Horder 

Str.  pyogenes.  . 

X 

X 

X 

o 

o 

X 

+ 

_ 

Str.  salicarius.  . 

X 

X 

X 

© 

o 

o 

— 

11 

Str.  anginosus.  . 

X 

X 

X 

© 

0 

0 

+ 

— 

" 

Str.  gracilis  .... 

X 

X 

0 

0 

X 

X 

- 

+ 

ii 

? 

X 

X 

o 

o 

X 

X 

— 

— 

" 

Str.  fsecalis  .... 

X 

X 

X 

0 

X 

X 

_ 

— 

" 

Str.  versatilis.  . 

X 

X 

X 

X 

X 

X 

— 

— 

Broadhurst 

Str.  bovinus.  .  . 

X 

X 

X 

X 

o 

X 

— 

— 

Winslow 

X  indicates  that  test  substance  is  fermented  with  production  of  acid  and  without 
gas  formation. 

©  indicates  that  test  substance  is  occasionally  fermented. 


156  PATHOGENIC  ORGANISMS 

The  fermentation  and  hsemolytic  reactions  of  the  best- 
known  types  of  streptococci,  excepting  Str.  lacticus,  are  shown 
in  Table  LIX. 

B.  DIPHTHERIA 

Milk  has,  on  several  occasions,  been  proved  to  be  a  vehicle 
for  B.  diphtheria  and  responsible  for  epidemics  of  diphtheria, 
and  it  is  consequently  sometimes  necessary  for  the  bacteriolo- 
gist to  examine  milk  for  this  organism. 

There  is  no  satisfactory  evidence  that  diphtheria  organisms 
may  invade  the  udder  and  so  cause  infection  of  the  milk,  but 
it  is  more  than  probable  that  milk  has  become  accidentally  in- 
fected from  human  sources  and  that  the  organisms  have  rapidly 
increased  in  number.  Milk  is  not  an  ideal  medium  for  the 
development  of  B.  diphtheria  but  fairly  rapid  multiplication 
does  occur  until  checked  by  the  metabolic  products  of  the  acid 
producers. 

The  number  of  authentic  cases  in  which  B.  diptherise  has 
been  isolated  from  milk  are  comparatively  few.  Bowhill,10 
in  1899,  isolated  diphtheria  organisms  from  milk  and  prepared 
broth  cultures  that  were  fatal  to  guinea  pigs  in  forty-eight  hours. 
The  same  year  Eyre  n  isolated  a  virulent  diphtheritic  bacillus 
from  milk  and,  later,  cases  were  reported  by  Klein,12  Dean  and 
Todd  13  and  Marshall.14 

For  the  isolation  of  the  organisms,  Bowhill  directly  inocu- 
lated Loeffler's  blood  serum  with  the  sample.  Eyre,  and  Dean 
and  Todd  concentrated  the  organisms  by  centrifugalising  and 
afterwards  streaked  the  sediment  over  a  number  of  tubes  of 
blood  serum.  The  cream  layer  was  treated  in  a  similar  man- 
ner. Characteristic  colonies  were  fished  and  those  mor- 
phologically resembling  B.  diphtheria  isolated  as  pure  cultures 
and  tested  for  pathogenicity.  Klein  and  Marshall  used  the 
animal  inoculation  method.  The  former  inoculated  two  guinea 
pigs  with  one  sample,  one  subcutaneously  in  the  groin,  and  the 
other  intraperitoneally.  The  latter  pig  remained  well,  but  the 
former,  on  the  fifth  day,  showed  swollen  inguinal  glands  sur- 


B.  DIPHTHERIA  157 

rounded  by  soft  cedematous  tissue.  On  autopsy  the  sub- 
cutaneous tissue  in  the  region  of  the  seat  of  inoculation  was 
cedematous  and  streaked  with  blood.  The  inguinal  glands 
were  enlarged,  firm,  and  deeply  congested.  Film  preparations 
from  the  juice  of  the  incised  gland  showed  numerous  diphtheritic 
organisms.  A  pure  culture  was  obtained  which  was  proved  to 
be  B.  diphtheria  by  the  virulence  test  and  also  by  the  antitoxin 
test. 

For  the  examination  of  milk  for  B.  diphtherias,  the  serum 
method  undoubtedly  offers  the  best  chance  of  obtaining  a  posi- 
tive result.  50  c.cms.  of  sample  are  centrifugalised  at  2000 
revolutions  per  minute  fof  twenty  minutes  and  the  cream  layer 
removed  to  a  sterile  dish.  The  milk  layer  is  withdrawn  by 
means  of  a  suction  pump  connected  to  a  fine  bore  glass  tube 
until  only  1-2  c.cms.  remain.  The  sediment,  and  cream 
layer,  are  used  for  inoculating  either  blood  serum  plates  or 
tubes.  If  tubes  are  used,  one  loopful  is  employed  for  smearing 
the  surface  of  a  number  of  tubes  in  succession  so  that  at  least 
one  tube  will  be  obtained  in  which  the  colonies  are  well  isolated. 
In  this  manner  a  total  of  from  40  to  50  tubes  is  used  for  one 
sample  and  examined  after  sixteen  or  eighteen  hours  incubation 
at  37°  C.  From  the  tubes  containing  well-isolated  colonies, 
subcultures  are  made  of  all  colonies  in  any  way  resembling  B. 
diphtherias  and  examined  as  to  their  morphological  character- 
istics and  biochemical  properties.  B.  diphtherias  is  usually 
found  in  fresh  serum  preparations  as  a  slender  rod  about  3ju  in 
length  and  exhibiting  well-defined  polar  granules  when  stained 
with  Loeffler's  methylene  blue  or  Ponder 's  stain  (see  appendix). 
The  club-shaped  bacillus  is  sometimes  found,  and  also  beaded 
and  barred  varieties  but  the  bipolar  type  (type  c,  Westbrook 
classification)  is  the  most  typical.  B.  diphtherias  does  not 
liquefy  gelatine,  is  Gram  positive,  and  ferments  dextrose, 
Isevulose,  galactose,  arabinose,  and  maltose  without  formation 
of  gas  but  not  saccharose  and  mannite.  Older  cultures  some- 
times produce  acid  in  lactose  and  glycerine.  The  bacillus  is 
non-motile  and  does  not  form  spores. 


158  PATHOGENIC  ORGANISMS 

The  organisms  that  pass  the  morphological  and  biochemical 
tests  must  be  tested  for  virulence  to  guinea  pigs.  Two  pigs 
are  used,  one  for  a  subcutaneous  or  intra-peritonial  injection  of 
the  twenty-four  hour  broth  culture  alone  (1  c.cm.)  and  the  other 
for  a  mixture  of  the  culture  with  1  c.cm.. of  a  diphtheritic  anti- 
toxin of  high  titre.  The  unprotected  pig  usually  dies  within 
thirty-six  hours,  and  almost  invariably  within  forty-eight  hours, 
if  the  culture  is  one  of  typical  B.  diphtherise.  The  protected 
animal  should  show  no  definite  symptoms  and  remain  alive. 

Diphtheroid  Bacilli.  On  many  occasions  bacilli  have  been 
found  in  milk  having  the  characteristic  granular  staining  prop- 
erties of  some  forms  of  B.  diphtherise  but  sharply  differentiated 
from  this  organism  by  the  absence  of  virulence.  Bergey15 
investigated  a  number  of  these  organisms  which  were  apparently 
identical  with  B.  diphtheria,  and  divided  them  into  three 
groups  according  to  their  biochemical  properties.  Two  groups 
showed  fermentative  activity  markedly  different  to  the  diph- 
theritic group  and  that  of  the  third  was  identical  but  non- 
pathogenic.  Savage 16  investigated  a  number  of  the  diph- 
theroid  organisms  found  in  milk  sediments.  These  were 
apparently  identical  and  closely  resembled  B.  diphtherise  in 
staining  properties  and  microscopical  appearance  except  for  an 
absence  of  blue  granules  in  preparations  stained  with  Neisser's 
stain.  The  bacilli  were  Gram  positive,  non-motile,  and  devel- 
oped on  nutrient  agar  as  small,  discrete,  translucent  colonies. 
On  serum  they  were  slightly  coloured  and  such  organisms  did 
not  give  the  typical  microscopical  appearance  found  with  the 
growths  on  agar.  Litmus  milk  was  unaffected  and,  except  for 
a  trace  of  acid  in  lactose,  neither  gas  nor  acid  was  produced 
in  the  usual  test  media.  They  were  non-pathogenic  to  mice. 
Klein  17  found  a  bacillus  in  milk  which  he  called  B.  diphther- 
oides.  This  organism  differed  morphologically  from  B.  diph- 
therise, Hoffmann's  bacillus,  and  the  xerosis  group.  No 
growth  was  observed  on  gelatine  at  21°  C.  or  on  agar  at  temper- 
atures less  than  25°  C.  On  agar  at  37°  C.  the  growth  was  slow 
and  no  colonies  appeared  until  the  third  day  when  they  devel- 


B.  TYPHOSUS  159 

oped  as  small  grey  dots.  Milk  was  coagulated  at  37°  C.  with 
acid  formation  and  a  separation  of  the  milk  constituents  into 
a  cream  layer  at  the  top,  curd  at  the  bottom,  and  whey  in 
between.  On  blood  serum  the  colonies  appeared  on  the  third 
day  as  depressions  due  to  liquefaction  of  the  medium.  On 
injection  into  guinea  pigs,  well-developed  local  abscesses  ap- 
peared in  one  to  two  weeks.  Intra-peritoneal  injection  pro- 
duced abscesses  on  the  omentum  and  on  the  pancreas  or  around 
the  kidney.  The  author  has,  on  several  occasions,  isolated 
bacilli  from  milk  that  resembled  B.  diphtheria,  but  the  majority 
of  these  could  be  distinguished  from  the  typical  pathogenic 
variety  by  the  size.  The  most  usual  type  was  about  5/*  in 
length  and  slightly  pointed  at  both  ends;  they  retained  the 
stain  when  treated  by  Gram's  method  and  gave  a  typical 
barred  appearance  with  Loeffler's  methylene  blue  and  Ponder's 
stain.  On  agar,  and  on  blood  serum,  the  organisms  developed 
as  small  white  opaque  colonies.  Gelatine  was  not  liquefied. 
Dextrose,  lactose,  saccharose,  mannite,  and  dulcite  were  not 
fermented  and  no  visible  change  was  produced  in  litmus  milk. 
They  were  non-motile  and  did  not  form  spores;  broth  cultures 
were  non-pathogenic  to  guinea  pigs  when  treated  by  the  intra- 
peritoneal  method, 

No  etiological  connection  has  been  discovered  between 
these  diphtheroid  bacilli  and  any  pathological  condition  and 
they  must,  therefore,  be  regarded  as  harmless  saphrophytes 
that  are  of  no  importance  or  significance  in  public  health  work. 

B.  TYPHOSUS 

There  are  on  record  several  hundreds  of  epidemics  of 
typhoid  fever  that  are  definitely  attributed  to  milk  as  the 
immediate  source  of  infection,  but  there  is,  so  far  as  the  author 
can  ascertain,  not  a  single  authentic  case  recorded  in  which  B. 
typhosus  has  been  isolated  from  milk  suspected  of  causing  an 
epidemic.  Typhoid  infection  of  milk  is  of  external  origin  and 
whether  it  is  due  to  a  carrier,  or  to  a  person  having  the  dis- 


160  PATHOGENIC  ORGANISMS 

ease,  or  water,  it  is  almost  invariably  intermittent  or  transitory 
with  the  consequence  that  by  the  time  an  outbreak  has  oc- 
curred and  can  be  traced  to  the  milk  supply  it  is  almost  hopeless 
to  expect  to  isolate  the  infecting  organism.  This,  however, 
should  not  deter  those  responsible  for  the  investigation  of  such 
cases  from  attempting  the  isolation  of  B.  typhosus. 

Isolation  of  B.  Typhosus.  Jackson  and  Melia18  recommend 
inoculating  the  sample  into  lactose  bile  and  incubating  at  37°  C. 
The  cultures  are  to  be  transplanted  in  varying  dilutions  into 
Hesse  agar  and  examined  after  twenty-four  hours  at  37°  C. 
On  this  medium  B.  coli  forms  small  succinct  colonies;  B. 
typhosus  is  most  characteristic  on  plates  containing  but  few 
colonies;  colonies  of  a  large  size  are  then  formed,  often  several 
centimetres  in  diameter,  and  consisting  of  a  broad  translucent 
or  scarcely  turbid  zone  between  a  white  opaque  centre  or  nucleus 
and  the  perfectly  circular  narrow  white  edge.  Tonney  et  al.19 
found  that  lactose  bile  is  inhibitory  to  B.  typhosus  as  well  as  to 
the  colon  group  of  organisms  and  this  is  confirmed  by  the  au- 
thor's experience. 

The  following  method,  which  is  an  adaptation  of  Browning 
and  Thornton's  method40  for  the  isolation  of  typhoid  bacilli 
from  faeces,  can  be  recommended  for  the  isolation  of  B.  typhosus 
from  milk.  Centrifugalise  50  c.cms.  of  the  sample  for  twenty 
minutes  at  2000  to  2500  revolutions  per  minute.  Remove  the 
cream  layer  to  a  sterile  tube  and  place  it  in  a  water  bath  at 
37°  to  40°  C.  Draw  off  the  skim  milk  by  means  of  a  fine  glass 
tube  attached  to  a  suction  pump  until  about  3  c.cms.  remain. 
After  thoroughly  distributing  the  sediment  throughout  the 
liquid  it  is  inoculated  into  three  brilliant  green  peptone^tubes, 
one  cubic  centimetre  being  placed  in  each  tube.  The  molten 
cream  layer  should  be  similarly  treated  as  a  proportion  of  the 
organisms  may  be  trapped  by  the  rising  fat  globules  during  the 
centrifugalising  process.  The  brilliant  green  medium  is  pre- 
pared by  steaming  a  2  per  cent  peptone  solution,  containing 
0.5  per  cent  of  sodium  chloride,  for  forty-five  minutes  and 
filtering  after  making  the  reaction  slightly  alkaline  to  litmus. 


GAERTNER  GROUP  161 

The  medium  is  sterilised  under  pressure  either  in  bulk  or  in 
10  c.cm.  quantities  in  tubes.  The  brilliant  green  (Hochst)  is 
kept  as  a  stock  1  per  cent  solution  which  is  made  into  a  1  in 
10,000  solution  just  before  use  by  diluting  0.1  c.cm.  to  10  c.cms. 
Before  inoculating  the  10  c.cms.  of  peptone  saline  medium  with 
the  suspected  material,  0.5  c.cm.  of  the  1  in  10,000  brilliant  green 
solution  is  added.  The  tubes  are  incubated  at  37°  C.  for  twenty 
to  twenty-four  hours  and  then  plated  out  on  neutral  red  bile  salt 
agar  or  Endo's  medium,  preferably  the  former.  The  colourless 
characteristic  colonies  are  fished  and  put  through  the  usual 
agglutination  and  biochemical  tests.  Using  this  method,  the 
author  has  been  able  to  isolate  B.  typhqsus  from  the  sediment  of 
milk  to  which  had  been  added  23  typhoid  bacilli  per  100  c.cms. 

Paratyphoid-enteritidis  or  Gaertner  Group.  The  organisms 
of  this  group  may  be  isolated  by  the  same  method  as  is  given 
above  for  B.  typhosus  or,  if  no  examination  is  required  for  B. 
typhosus,  the  sediment  and  cream  may  be  inoculated  into  meat 
peptone  dextrose  broth  (neutral  to  phenolphthalein)  containing 
0.15  c.cm.  of  a  1  per  cent  solution  of  brilliant  green  per  10  c.cms. 
of  broth.  (Tonney.20)  This  strength  of  brilliant  green  (1  in 
6600)  inhibits  the  growth  of  the  Escherich  and  Eberth  groups, 
and  enables  the  Gaertner  group  to  predominate  the  broth  cul- 
tures. The  broth  cultures  are  subsequently  plated  out  on 
neutral  red  lactose  bile  salt  agar  and  the  non-lactose  fermenters 
worked  out  in  the  usual  way. 

Morgan's  Bacillus  No.  i.  During  the  last  few  years  the 
attention  of  sanitarians  has  been  directed  to  the  etiological 
relationship  between  milk  supplies  and  epidemic  summer 
diarrhoea.  It  has  been  evident  for  many  years  that  artificial 
feeding  of  infants  was  a  contributing  factor  but  no  definite 
cause  was  assigned  for  this  phenomenon.  Defective  feeding  has, 
no  doubt,  contributed  to  the  excessive  infantile  mortality  that 
occurs  each  summer,  but  there  is  a  rapidly  accumulating  mass 
of  evidence  that  the  epidemic  variety  of  summer  diarrhoea  is 
primarily  or  secondarily  dependent  upon  the  activity  of  micro- 
organisms. The  substitution  of  a  clean  milk  supply  or  the 


162  PATHOGENIC  ORGANISMS 

pasteurisation  of  the  old  supply  has,  in  many  cases,  led  to  an 
abatement  of  infantile  diseases  and  this  would  indicate  that  an 
excessive  number  of  bacteria  of  all  kinds  and  not  any  particular 
group  is  responsible  for  the  effects  observed.  (Park  and  Holt.21) 

Scholberg  and  Wallis22  suggest  that  the  prejudicial  effect 
is  due  to  physical  and  chemical  changes  produced  by  bacterial 
contamination.  They  found  that  the  products  of  proteoclastic 
digestion  appear  in  milk  as  the  atmospheric  temperature  in- 
creased and  that  the  albumoses  and  peptones  so  produced  may 
be  toxic  to  infants. 

Morgan  and  Ledingham,23  in  1909,  made  an  investigation 
of  the  bacteriology  of  summer  diarrhsea  and  concluded  that  a 
non-lactose  fermenting,  non-liquefying  organism  which  they 
isolated  and  which  is  now  usually  known  as  Morgan's  Number  1 
Bacillus,  bore  a  close  relationship  to  the  disease. 

Lewis,24  Ross,25  O'Brien  26  and  Orr,27  made  numerous  exami- 
nations of  the  faeces  of  infants  and,  although  they  found  that 
the  non-gelatine  liquefying,  non-lactose  fermenters  were  ab- 
normally prevalent  in  the  cases  of  diarrhea,  they  could  not 
establish  any  definite  causal  relationship.  In  1911,  Lewis28 
and  Alexander29  made  further  observations  on  this  group  and 
showed  that  Morgan's  No.  1  Bacillus  was  conspicuously  fre- 
quent in  the  faeces  of  infants  having  epidemic  diarrhoea.  In 
the  same  year  Graham  Smith30  found  that  the  non-gelatine 
liquefying  non-lactose  fermenters  were  especially  prevalent 
in  flies  during  the  seasonal  prevalence  of  diarrhoea  and  that 
Morgan's  No.  1  Bacillus,  whilst  rarely  present  in  flies  from 
houses  not  containing  diarrhoeal  cases,  was  frequently  found  in 
houses  associated  with  this  disease. 

Lewis  31  pointed  out  the  importance  of  applying  the  agglu- 
tination test  to  the  various  organisms  which  gave  the  usual 
fermentation  reactions  for  Morgan's  No.  1  Bacillus. 

The  etiological  relationship  of  Morgan's  No.  1  Bacillus  to 
epidemic  summer  diarrhoea  is  not  yet  fully  established,  but  the 
evidence  in  favour  of  this  hypothesis  is  undoubtedly  strong  and 
points  to  the  infection  of  the  milk  supply  in  the  home  by  flies. 


COLI-TYPHOID  GROUP 


163 


3333 

'S'3'3'3 


3  3  '3  '3 


'3333   ' 
!*S*0*S"3    ! 


1    1    1  -H  1  + 


1   1  -H  1 


++++  1  + 


XIII 


+++  1  +  1 


X  1  X  1 


++  1  1  1 


+++  1  +  1 


1    1    1    1 


++  1 


1  +  1 


Mill 


i  I   i   i   I   I 


+ 


1  X|X|  1 


XIII 


1  +-H  1  + 


1  1  1 


1   1   1   1 


1  ++  1  + 


1  1  1  1 


X  1  X  1 


XXX  1 


XXX  1 


XXX  1 


II 
X 

g 

I 

II 

F"^     CQ 

i  M 

i! 

^73 
O  fl 

1^) 

63 


1  1  1  1  + 


++-H-H  + 


g-.S-Sg   :   •     a 

*Ifli :-  iiinii 

iiijii  iiiiiii 

^  8  §^  S'w    ^gSaa'owg 

to  PQ  PQ  PQ  PQ  PQ      <u  PQ  PQ  pq  pq  pq  ^      «  pq"  pq'  pq  pq' 


1111 

rf>  o  a  a 
* 


164  PATHOGENIC  ORGANISMS 

Examination.  Morgan's  No.  1  Bacillus  is  very  suscep- 
tible to  the  action  of  brilliant  green  and  will  not  appear  on  the 
rebipelagar  plates  in  the  enrichment  method  for  isolating  the 
organisms  of  the  Gaertner  group.  The  best  procedure  is  to 
inoculate  the  centrifugalised  deposit  from  about  40-50  c.cms. 
of  milk  into  a  number  of  tubes  of  neutral  red  lactose  bile  salt 
agar  and  incubate  for  twenty-four  to  forty-eight  hours  after 
mixing  and  pouring  into  petri  plates.  All  colourless  colonies 
are  fished  into  dextrose  broth  and  those  organisms  producing 
acid  and  gas  in  this  medium  are  afterwards  tested  in  the  usual 
media  for  biochemical  reactions  and  also  with  a  specific  serum 
in  low  dilution.  Morgan's  No.  1  Bacillus  invariably  ferments 
dextrose  and  laevulose,  and  usually  also  arabinose  and  galactose, 
with  the  production  of  acid  and  gas.  Mannite  is  also  usually 
fermented  but  not  saccharose,  dulcite,  maltose,  dextrin,  or 
salicin.  Indol  is  produced  in  peptone  water  and  milk  becomes 
alkaline  in  about  ten  days.  Gelatine  is  not  liquefied. 

B.  TUBERCULOSIS 

For  the  detection  of  B.  tuberculosis  in  milk  two  processes 
have  been  employed :  (a)  the  microscopical  and  (6)  the  inocula- 
tion method. 

Microscopical.  In  very  rare  cases  the  presence  of  B.  tuber- 
culosis in  milk  may  be  demonstrated  by  the  examination  of 
stained  films  of  the  milk  without  previous  concentration,  but 
the  percentage  of  positive  results  so  obtained  is  so  small  as  to 
render  the  process  valueless  for  public  health  work.  When 
the  organisms  are  comparatively  numerous  they  may  be  found 
in  the  deposit  obtained  by  centrifugalising  50  to  100  c.cms.  of 
milk  at  2000  to  3000  revolutions  per  minute  for  thirty  minutes. 
For  the  preparation  of  cover-slip  films  Delepine  32  recommends 
spreading  small  portions  of  the  sediment  over  cover  slips  which, 
when  dry,  are  placed  in  a  covered  capsule  containing  equal 
parts  of  absolute  alcohol  and  ether  for  two  hours.  At  the 
expiration  of  this  period  the  capsule  is  placed  in  a  dish  con- 


INNOCULATION  METHOD  165 

taining  water  at  80°  to  90°  C.  The  mixture  of  alcohol  and 
ether  boils  at  once  and  after  ten  to  fifteen  minutes  the  cover 
slips  are  removed  and  washed  with  absolute  alcohol.  The 
films  are  then  stained  with  carbol-fuchsine  and  counterstained 
with  methyline  blue  according  to  the  Ziehl-Neelson  method 
which  is  as  follows: 

(1)  Stain  in  hot  carbol-fuchsine  for  five  to  ten  minutes,  being 
careful  to  avoid  over-heating. 

(2)  Decolourise  by  dipping  in  25  per  cent  sulphuric  acid. 

(3)  Wash  in  water. 

(4)  Wash  in  alcohol  until  no  more  stain  is  removed. 

(5)  Wash  in  water. 

(6)  Counterstain  for  one  minute  with  methylene  blue. 

(7)  Wash  in  water,  dry,  and  mount. 

Delepine  found  that  when  this  method  of  preparation  was 
carefully  followed,  very  clear  films  were  obtained  and  no  dif- 
ficulty was  caused  by  other  acid  fast  bacilli  when  sufficient 
attention  was  paid  to  the  morphological  characteristics  of  the 
organisms. 

Inoculation  Method.  The  inoculation  method  is  the  only 
one  that  can  be  relied  upon  for  the  detection  of  very  small 
numbers  of  B.  tuberculosis  in  milk,  but  the  time  required  to 
obtain  reliable  results  is  not  less  than  three  weeks  as  com- 
pared with  the  few  hours  required  for  the  completion  of  the 
microscopical  method.  It  is  good  routine  practice  to  make 
microscopical  preparations  of  all  sediments  obtained  by  cen- 
trifugalisation  and  to  inoculate  those  yielding  negative  or  doubt- 
ful results. 

To  prepare  the  sediment,  100  c.cms.  of  milk  are  centrifu- 
galised  at  2000  to  3000  revolutions  per  minute  for  at  least 
thirty  minutes,  and,  after  removing  the  cream  layer  with  a 
sterile  spatula  or  spoon,  the  separated  milk  is  drawn  off  through 
a  small  bore  glass  tube  attached  to  a  suction  pump  until  about 
4  c.cms.  of  milk  remain.  This  milk  is  thoroughly  mixed  with 
the  deposit  and  subsequently  used  for  the  inoculation  of  two 
animals.  If  the  milk  is  known  to  be  "  clean  "  the  milk  may 


166  PATHOGENIC  ORGANISMS 

be  reduced  to  2  c.cms.  and  only  one  animal  used  for  the  deposit, 
the  other  being  reserved  for  a  portion  of  the  cream  layer. 

On  account  of  its  sensitiveness  to  tuberculosis,  the  guinea 
pig  is  the  most  suitable  animal  for  inoculation  and  the  best 
results  are  obtained  with  animals  weighing  from  200  to  300 
grams. 

Two  methods  of  inoculation  are  in  general  use:  (a)  subcuta- 
neous injection  at  the  inner  side  of  the  left  hind  leg  and  (6) 
intraperitoneal  injection  through  the  belly  wall.  Delepine 
prefers  to  inoculate  at  the  inner  aspect  of  the  left  leg  at  the  level 
of  the  femoro-tibial  articulation  on  account  of  the  comparative 
results  obtained  by  the  uni-lateral  development  of  the  lesions. 
This,  he  found,  was  especially  noticeable  in  the  early  stages 
with  small  amounts  of  infectious  material  and  by  noting  the 
extent  of  the  lesion  development  in  two  pigs  killed  after  twenty- 
one  and  thirty-five  days,  a  rough  estimation  of  the  degree  of 
infectivity  was  procured.  In  the  very  early  stages  the 
lesions  were  limited  to  the  subcutaneous  tissue  and  the 
four  groups  of  lymphatic  glands  (the  popliteal,  superficial 
inguinal,  deep  inguinal,  and  the  sacro-lumbar)  on  the  same 
side  of  the  body  as  the  seat  of  inoculation.  Later  the  retro- 
hepatic  gland  and  spleen  were  involved  followed  by  the  liver, 
lungs,  bronchial  suprascapular,  and  cervical  glands  on  both 
sides  of  the  body.  Finally  there  was  a  more  complete  invasion 
of  the  lymphatic  glands  in  front  of  the  diaphragm  on  both  sides 
of  the  body  and  an  involvement  of  the  superficial  and  deep 
inguinal  and  other  glands  behind  the  diaphragm  on  the  right 
side  of  the  body. 

With  the  intra-peritoneal  inoculation  the  lymphatic  glands 
of  the  peritoneum  and  mysentery  are  first  involved,  followed 
by  the  liver  and  spleen.  The  cervical,  bronchial,  inguinal,  and 
popliteal  follow,  but  the  lesion  development  is  bilateral  through- 
out. 

In  order  to  accelerate  the  development  of  the  disease  when 
the  subcutaneous  method  is  used,  Block  33i  suggested  that  the 
inguinal  glands  on  the  inoculation  side  should  be  slightly  dam- 


INNOCULATION  METHOD  167 

aged  by  squeezing  them.  This  procedure  reduces  the  resistance 
of  the  glands  and  enables  an  earlier  diagnosis  to  be  made. 
Dodd,34  and  Joannovico  and  Kapsammer35  carefully  studied 
this  technique  and  found  it  entirely  successful.  They  found 
that  even  doubtful  cases  could  be  diagnosed  within  fourteen 
days. 

The  microscopic  appearance  of  the  lesions  is  usually  suf- 
ficient to  enable  a  trained  observer  to  make  an  accurate  diag- 
nosis, but  in  all  doubtful  cases  cover  slips  preparations  should  be 
made  and  supplemented  if  necessary  by  histological  sections. 
For  cultures,  nodules  are  squeezed  between  two  sterile  slides 
and  the  contents  smeared  over  glycerine  agar  slopes.  The 
cultures  are  incubated  at  37°  C. 

For  the  differentiation  of  tubercular  from  other  infections, 
Anderson  36  suggested  the  subcutaneous  injection  of  2  c.cms. 
of  tuberculin.  In  a  healthy  animal  a  slight  febrile  reaction 
occurs  and  passes  off  in  a  few  hours,  but  this  quantity  of  tuber- 
culin is  sufficient  to  cause  death  in  less  than  twenty-four  hours  in 
a  guinea  pig  showing  well  developed  tuberculosis.  When  the 
lesions  are  slight  the  animal  will  become  sick  but  may  not  die. 
This  method  may  be  used  as  an  addition  to  the  usual  autopsy 
but  should  not  be  substituted  for  it. 

Even  when  the  best  technique  is  used,  it  is  often  found  that 
the  experimental  animals  may  die  from  acute  infections  within 
a  few  days  of  inoculation.  This  is  due  to  "  dirty  "  milk  and 
can  be  partially  eliminated  by  the  treatment  of  the  sediment 
with  5  per  cent  antiformin  for  thirty  minutes  and  finally 
washing  with  physiological  saline.  Eastwood  and  Griffith37 
found  that  10  per  cent  antiformin  slightly  weakened  the  tubercle 
bacilli  and  that  a  20  per  cent  solution  almost  destroyed  them. 

Death  of  the  inoculated  animals  after  ten  days,  from  infec- 
tions other  than  generalised  tuberculosis,  is  largely  due  to 
improper  attention  to  the  housing  conditions  of  the  guinea 
pigs.  These  must  be  kept  isolated  in  clean  cages  with  not  more 
than  two  animals  to  a  cage  and  housed  in  well-ventilated 
rooms. 


168  PATHOGENIC  ORGANISMS 

Pseudo-tuberculosis.  Milk  occasionally  contains  organisms 
capable  of  producing  chronic  lesions  which  partially  simulate 
those  of  B.  tuberculosis  and  to  which  the  designation  of  pseudo- 
tuberculosis  has  been  given.  Delepine  found  that  amongst 
these  infections  was  one  resembling  chronic  pyaemia,  but  in  his 
opinion  the  resemblance  is  superficial  and  no  experienced  pathol- 
ogist could  mistake  such  lesions  in  the  guinea  pig  for  true 
tuberculous  lesions;  also  that  an  experimenter  with  scanty 
pathological  experience  could  not  make  a  mistake  if  the  or- 
ganisms in  the  lesions  are  microscopically  examined.  The 
finding  of  the  giant  cells,  characteristic  of  true  tuberculosis,  in 
histological  sections  would  also  clear  up  doubtful  microscopic 
diagnoses. 

In  pigs  that  have  been  kept  for  five  to  six  weeks  the  chronic 
lesions  due  to  B.  abortus  may  be  found,  but  as  this  organism  is 
not  acid  fast  there  is  no  difficulty  in  eliminating  this  possible 
source  of  error. 

Bovine  and  Human  Types  of  B.  Tuberculosis.  The  differ- 
ence in  the  cultural  and  other  characteristics  of  these  types  is 
essentially  relative  rather  than  absolute  and  this  fact  must 
always  be  kept  in  mind  when  attempting  to  classify  cultures  of 
B.  tuberculosis. 

Eastwood  and  Griffith38  classified  cultures  as  dysgonic  or 
eugonic  according  to  the  luxuriance  of  the  growth  on  glycer- 
inised  agar  and  they  found  that  the  dysgonic  type  was  usually 
of  high  virulence  for  rabbits  and  corresponded  to  the  bovine 
type.  The  human  type  grew  well  on  glycerine-agar  but  pos- 
sessed much  lower  virulence  for  rabbits.  The  chief  differences 
in  the  two  types  may  be  summarised  as  follows : 

BOVINE.  HUMAN. 

Morphology.  Only  slight  differences  can  be  found,  the  bovine  organisms 
being  usually  shorter,  straighter,  and  thicker. 

Cultural  characteristics. 

Glycerine-agar.  Grows  feebly  and  Grows  luxuriantly  and  usually 
with  development  of  discrete  col-  without  difficulty.  Growth 

onies.  often  wrinkled. 


BIBLIOGRAPHY  169 

Bovine  serum.     Grows  slowly  and  Grows  fairly  rapidly. 

appears  as  a  fine,  filmy,  non-pig- 

mented  growth  after  two  to  three 

weeks. 
Glycerine   broth   2    per   cent   acid. 

Acid  reaction  diminishes  and  may        Remains  permanently  acid. 

finally  become  alkaline. 
Pathogenicity. 

Calves.     Highly  pathogenic.  Non-pathogenic. 

Rabbits.     Highly  pathogenic.  Slightly  pathogenic.     The  lesions 

Subcutaneous  inoculation  with  10        are  often  localised  in  the   lungs 
m.gr.  causes  an  acute  generalised        and  kidneys  or  scattered, 
fatal  tuberculosis. 

In  the  preparation  of  cultures  from  lesions  for  differentiation 
of  type  the  primary  ones  should  be  made  on  Dorset's  egg  medium 
(see  Appendix)  and  subcultivated  to  blood  serum  or  glycerine- 
agar. 

BIBLIOGRAPHY 

1.  Coleman.     Rpt.  of  M.  O.  for  Angelsey.     1897. 

2.  Savage,     Rpt.  of  M.  O.  to  L.  G.  B.    1906-07,  228-252,  ibid.,  1907-08, 

359-424,  ibid.,  1908-09,  294-315. 

3.  Krumwiede  and  Valentine.     Rpt.  36  New  York  City  Health  Dept. 

4.  Jackson.     Jour.  Inf.  Dis.     1913,  12,  364-385. 

5.  Davis  and  Capps.     Jour.  Inf.  Dis.     1914,  15,  135-140. 

6.  Heinemann.     Jour.  Inf.  Dis.     1906,  3,  175. 

7.  Heinemann.     Jour.  Inf.  Dis.     1907,  4,  87-92. 

8.  Muller.     Arch,  f .  Hyg.     1906,  56,  90. 

9.  Heinemann.     Jour.  Inf.  Dis.     1915,  16,  221-240. 

10.  Bowhill.     Jour.  State  Med.     1899,  705-710. 

11.  Eyre.     Brit.  Med.  Jour.     1899,  2,  586. 

12.  Klein.     Jour,  of  Hyg.     1901,  1,  85. 

13.  Dean  and  Todd.     Jour,  of  Hyg.     1902,  2,  194-205. 

14.  Marshall.     Jour,  of  Hyg.     1907,  7,  32. 

15.  Bergey.     Jour.  Med.  Research.     1904,  11,  445. 

16.  Savage.     Rpt.  of  M.  O.  to  L.  G.  B.     1906-07,  224-225. 

17.  Klein.     Jour,  of  Hyg.     1901,  1,  78. 

18.  Jackson  and  Melia.     Jour.  Inf.  Dis.     1909,  6,  194. 

19.  Tonney  et  al.     Jour.  Inf.  Dis.     1916,  18,  243. 

20.  Tonney.     Jour.  Inf.  Dis.     1913,  13,  263-272. 

21.  Park  and  Holt.     Arch,  of  Ped.     1913,  20,  881. 


170  PATHOGENIC  ORGANISMS 

22.  Scholberg  and  Wallis.     Rpt.  of  M.  O.  to  L.  G.  B.     1909-10,  504. 

23.  Morgan  and  Ledingham.     Proc.  Roy.  Soc.  Med.     2,  1909,  133. 

24.  Lewis.     Rpt.  of  M.  O.  to  L.  G.  B.     1910-11,  346. 

25.  Ross.     Rpt.  of  M.  O.  to  L.  G.  B.     1910-11,  366. 

26.  O'Brien.     Rpt.  of  M.  O.  to  L.  G.  B.     1910-11,  373. 

27.  Orr.     Rpt.  of  M.  O.  to  L.  G.  B.     1910-11,  386. 

28.  Lewis.     Rpt.  of  M.  O.  to  L.  G.  B.     1911-12,  286. 
29. 'Alexander.     Rpt.  of  L.  G.  B.     1911-12,  303. 

30.  Graham  Smith.     Rpt.  of  M.  O.  to  L.  G.  B.     1911-12,  319. 

31.  Lewis.     Rpt.  of  M.  O.  to  L.  G.  B.     1912-13,  375. 

32.  Detepine.     Rpt.  of  M.  O.  to  L.  G.  B.     1908-09,  370. 

33.  Bloch.     Berlin,  klin.  Wochenschrift.     1907,  40,  511. 

34.  Dodd.     Jour.  Roy.  Inst.  Pub.  Health.     1909,  17,  360. 

35.  Joannovico   and  Kapsammer.     Berlin,   klin.   Wochenschrift.     1907, 

44,  1439. 

36.  Anderson.     U.  S.  A.,  P.  H.  and  M.  H.  S.,  Hyg.  Lab.  Bull.  46,  183. 

37.  Eastwood  and  Griffiths.     Rpt.  of  M.  O.  to  L.  G.  B.     1912,  303. 

38.  Eastwood  and  Griffiths.     Rpt.  to  L.  G.  B.,  Pub.  Health  Series,  No.  88. 

39.  Winslow.    Jour.  Inf.  Dis.,  1912,  10,  285. 

40.  Browning  and  Thornton.     Brit.  Med.  Jour.     1915  (Aug.  14),  248-250. 

41.  Ruediger.    Science.     1912,  35,  223. 


CHAPTER  XIII 
CELLS,  DIRT  AND  DEBRIS 

Cells.  For  nearly  a  century  it  was  recognised  that  cells 
or  cell  fragments  were  present  in  the  secretion  as  formed  in 
the  alveoli,  but  it  is  only  comparatively  recently  that  any 
efforts  were  made  to  ascertain  if  any  cells  were  present  in  the 
discharged  milk.  In  1897  Stokes  and  Wegefarth 1  directed 
attention  to  the  presence  of  leucocytes  in  milk  and,  since  then, 
considerable  study  has  been  given  to  this  subject.  These 
observers  differentiated  the  leucocytes  from  the  epithelial  cells 
by  the  form  of  the  nuclei  but,  unfortunately,  designated  the 
former  as  pus  cells,  a  nomenclature  that  was  perpetuated  by 
many  later  writers.  This  designation  is  no  longer  accepted 
and  the  cells  are  regarded  as  constituents  of  normal  milk.  There 
is  still  some  diversity  of  opinion  regarding  the  nature  of  these 
cells,  some  experimenters,  including  Winkler,  Hewlett,  Villar, 
and  Revis,  holding  that  they  are  predominantly  of  epithelial 
origin,  whilst  others,  amongst  whom  are  Bergey,  Doane,  Miller, 
Breed,  Ernst,  and  Savage,  regard  them  mixtures  of  blood  cells 
and  epithelial  cells. 

Hewlett,  Villar,  and  Revis  2  support  the  contention  of  Wink- 
ler and  Michaelis  that  the  cells  in  normal  milk  are  chiefly  young 
epithelial  cells  which  have  become  detached.  In  a  later  paper 
they  find  that  in  the  milk  of  healthy  cows  in  full  milk  and 
which  do  not  give  a  high  cell  count,  the  majority  of  the  cells 
appear  to  be  "  large  uninuclears  "  with  a  small  admixture  of 
other  cells.  At  the  beginning  and  end  of  lactation  and  when 
the  cell  count  was  high  from  other  causes,  whether  physiological 
or  pathological,  the  "  multinu clears  "  predominated.  Scan- 
nel 3  pointed  out  that  epithelial  cells  are  mononuclear  and  that, 

171 


172  CELLS,  DIRT  AND  DEBRIS 

although  on  dividing,  they  may  appear  as  polymorphonuclears 
it  is  inconceivable  that  they  should  divide  at  such  a  rate  as  to 
produce  500,000  per  c.cm.  There  are  also  certain  histological 
characteristics  that  differentiate  nucleated  epithelial  cells  and 
mononuclear  leucocytes. 

The  views  of  those  who  regard  the  cells  found  in  milk  as 
mixtures  of  blood  and  epithelial  cells,  which  is  the  more  gen- 
erally accepted  explanation,  are  well  set  forth  in  a  recent  book 
by  Ernst 4  in  which  the  histological  characters  of  the  cells  are 
treated  "  in  extenso." 

According  to  Ernst  the  cells  are  of  dual  origin,  (a)  Epi- 
thelial cells  derived  from  the  tissue  lining  the  ducts  and  from  the 
secretory  glands  and, 

(6)  Leucocytes  which  have  passed  through  the  walls  of  the 
capillaries  and  lymphatics  and  finally  obtained  access  to  the 
gland  secretion.  This  would  appear  to  be  normal  process  in 
all  secretory  glands.  Under  special  stimulation,  either  from 
mechanical  or  pathological  causes,  the  number  and  nature  of 
the  cells  may  undergo  radical  changes  depending  upon  the 
nature  and  extent  of  the  stimulation.  .  This  affords  a  rational 
explanation  of  the  diversified  cells  found  in  milk  and  alterations 
in  their  relative  proportions  under  varying  conditions.  A 
general  description  of  the  cells  usually  found  in  milk  follows. 

Epithelial  Cells,  (a)  From  compound  epithelium:  these 
are  found  as  small  platelets  often  folded  in  so  many  various  ways 
that  the  original  shape  of  the  cell  is  entirely  obscured.  They 
are  most  numerous  during  the  early  period  of  the  lactation  and 
are  due  to  the  mechanical  stimulation  of  the  teats  by  milking. 

(6)  From  the  milk  cistern;  usually  oval  or  rectangular  in 
shape,  frequently  elongated  to  a  point  along  the  longitudinal 
axis  and  having  an  oval  nucleus.  In  normal  milk  they  are 
usually  found  singly  but  increased  desquamation  produced 
by  stimulation  may  cause  masses  of  cells  to  appear  arranged  like 
the  petals  of  a  flower  round  a  common  centre. 

(c)  From  secretory  ducts  and  alveoli:  these  vary  in  size  accord- 
ing to  the  number  of  fat  globules  they  contain  (5  to  45  //)  and 


BLOOD  CELLS  173 

when  very  distended  they  are  known  as  "  foam  cells."  The 
nucleus  is  usually  well  marked  when  unmixed  with  fat  and  only 
surrounded  with  a  narrow  margin  of  protoplasm ;  the  presence 
of  fat  produces  the  characteristic  honeycombed  appearance  of 
the  colostral  bodies  and  such  cells  are  only  found  in  patho- 
logical conditions  and  at  the  beginning  and  end  of  the  lacta- 
tion period.  Some  observers  report  that  these  large  cells  may 
contain  several  nuclei,  but  Ernst  never  found  more  than  one 
and  suggested  that  the  apparent  multiplication  of  nuclei  was 
due  to  mononuclear  cells  becoming  superimposed. 

Blood  Cells,  (a)  Red  blood  cells  or  erythrocytes  appear  as 
biconcave  discs  or  as  thorn-apple  shaped  cells  containing  meta- 
chromatic  granules. 

(b)  Leucocytes.  These  constitute  a  very  considerable  per- 
centage of  the  total  cells  in  normal  physiological  conditions 
and  may  entirely  predominate  in  pathological  ones.  All 
varieties  of  leucocytes  may  be  found  but  the  usual  frequency 
of  occurrence  is  in  the  following  order:  polymorphonuclears, 
lymphocytes,  large  mononuclears,  and  transitionals. 

The  polymorphonuclear  leucocytes,  of  which  the  majority 
are  neutrophylic  in  their  staining  properties,  are  usually  7.5  to 
10  M  in  diameter  and  stain  characteristically  with  methylene 
blue  as  a  deeply  stained  lobed  or  polymorphonucleus  sur- 
rounded by  faintly  coloured  protoplasm.  The  lymphocytes  are 
usually  considerably  smaller  (5.7  /x)  than  the  "  polymorphs  " 
but  vary  very  considerably  in  size.  The  nucleus  is  round  and 
occupies  practically  the  whole  of  the  cell.  Mononuclear  leu- 
cocytes are  much  larger  than  the  lymphocytes  (usually  13-16  ju 
but  may  be  25  M  in  diameter)  and  two  to  three  times  the  size  of 
erythrocytes.  The  nucleus  is  large  and  oval  and  is  eccentrically 
situated  in  a  relatively  large  amount  of  protoplasm.  With 
methylene  blue  the  nucleus  stains  moderately  well  and  the 
cytoplasm  contains  fine  amorphous  particles  which  produce 
the  appearance  of  ground  glass.  With  Leishmann's  stain  the 
nucleus  is  ruby  coloured  and  the  cytoplasm  blue  but  containing 
a  few  ruby  granules.  The  transitional  cells  are  about  the  size  of 


174  CELLS,  DIRT  AND  DEBRIS 

the  large  mononuclears.  The  nucleus  shows  varieties  of  transi- 
tion between  the  indented  mononuclear  and  the  irregular  poly- 
morphonuclear  cell.  As  a  rule,  it  is  indented,  crescrentic  in 
shape,  and  not  possessing  the  multiplication  so  characteristic 
of  the  polymorphonuclear  leucocytes. 

Degenerated  cells  of  various  kinds  may  also  be  present  in 
milk.  Cells  may,  under  various  influences,  become  partially 
or  wholly  disintegrated  and  the  contents  dispersed  in  fragments. 
The  nucleus  may  split  up  and  the  chromatin  spread  through  the 
plasma  as  dust  or  flakes.  These  flakes  are  often  designated  as 
"  Nissen's  Globules  "  and  present  the  appearance  of  a  darkly 
stained  centre,  with  or  without  a  lightly  stained  border.  The 
albuminophores  of  Bab  and  Shulz  which  they  describe  as  lym- 
phocytes (15  to  20  M),  containing  fat  and  one  to  four  proteid 
bodies,  are  regarded  by  Ernst  as  degenerated  fat  containing 
cells  which  have  been  attacked  by  macrocytes  and  then  further 
degenerated  until  the  nucleus  is  no  longer  visible. 

Estimation  of  Cells.  The  first  attempt  to  estimate  the 
number  of  cells  in  milk  was  that  of  Stokes  and  Wegefarth  in 
1897  l  and  consisted  in  the  examination  under  an  oil  immersion 
lens  of  a  stained  film  prepared  from  the  sediment  obtained  by 
centrifugal  action.  This  method  was  adopted  with  but  slight 
modifications  by  Bergey,  Stewart  and  Slack. 

Doane  and  Buckley  in  1905 5  devised  what  is  known  as 
the  "  volumetric  method  "  in  which  a  counting  cell,  such  as  is 
commonly  used  in  the  estimation  of  cells  in  blood,  was  used  for 
the  enumeration  of  the  cells  in  the  centrifugalised  deposit  from 
10  c.cms.  of  milk.  Russell  and  Hoffmann 6  compared  the 
"  smeared  sediment  "  and  "  volumetric  "  methods  and  found 
an  average  variation  of  112  per  cent  in  the  former  as  against 
only  6  per  cent  in  the  latter.  They  found  also  7  that  a  pre- 
liminary heating  of  the  milk  to  70°  C.  produced  higher  and  more 
consistent  results.  The  details  of  this  method,  as  adopted  by 
the  Committee  on  Standard  Methods  of  Bacterial  Milk  Analysis 
of  the  American  Public  Health  Association  8  are  as  follows: 

Collection  of   Samples.     Samples  for  analysis  should   be 


CONCENTRATION  OF  CELLULAR  ELEMENTS         175 

taken  from  the  entire  milking  of  the  animal,  as  the  strippings 
contain  a  somewhat  larger  number  of  cells  than  other  portions 
of  the  milk.  For  the  purpose  of  examination  take  200  c.cms. 
in  a  stoppered  bottle. 

Time  Interval  between  Collection  and  Analysis.  To  secure 
satisfactory  results,  milk  must  be  examined  in  a  sweet  condi- 
tion. Development  of  acidity  tends  to  precipitate  casein 
in  the  milk  and  thus  obscure  the  examination  of  microscopic 
preparations.  Samples  received  from  a  distance  can  be  pre- 
served for  satisfactory  microscopical  examination  by  the 
addition  of  formalin  at  the  time  of  collection — a  proportion  of 
1  c.cm.  to  250  c.cms.  of  milk.  Formalin  has  been  found  the 
best  preservative  to  use  although  it  causes  contraction  of  the 
cells  to  some  extent. 

PROCEDURE  WITH  REFERENCE  TO  PREPARATION  OF  SAMPLE 

1.  Heating  Sample.     To  secure  the  complete  sedimentation 
of  the  cellular  elements  in  the  milk,  it  is  necessary  to  heat  the 
same  to  a  temperature  which  will  break  down  the  fat  globule 
clusters,  or  lessen  the  ordinary  creaming  properties  of  the  milk. 
Samples  should  be  heated  at  65°  to  70°  C.  for  not  less  than  ten 
minutes,  or  from  80°  to  85°  where  very  short  periods  of  exposure 
(one  minute)  are  given.     This  treatment  causes  the  more  homo- 
geneous distribution  of  the  fat  globules  through  the  milk,  and 
when  the  sample  is  then  subjected  to  centrifugal  force,  the 
cell  elements  are  not  caught  in  the  rising  fat  globules,  but  on 
account  of  their  higher  specific  gravity  are  concentrated  in  the 
sediment  by  centrifugal  force. 

2.  Concentration  of  Cellular  Elements.    After  centrifugali- 
sation  the  cream  and  the  supernatant  milk  are  removed,  with 
the  exception  of  the  last  \  c.cm.,  by  aspirating  with  an  exhaust 
pump  and  wiping  the  walls  of  the  tube  with  a  cotton  swab. 
After  thoroughly  mixing  the  sediment  with  a  glass  rod,  enough 
of  the  emulsion  is  placed  in  an  ordinary  blood  counter  (Thoma- 
Zeiss  pattern)  to  fill  exactly  the  cell.     The  preparation  is  then 
allowed  to  stand  for  a  minute  or  two  to  permit  the  cellular 


176  CELLS,  DIRT  AND  DEBRIS 

elements  to  settle  to  the  bottom  of  the  cell  while  the  few  fat 
globules  in  the  liquid  rise  to  the  surface.  This  method  permits 
of  the  differentiation  of  the  cells  from  the  small  fat  globules  in 
the  liquid  so  that  a  distinct  microscopic  observation  can  be 
made. 

Examination  of  Material.  The  preparation  is  examined 
in  an  unstained  condition.  The  count  is  made  with  a  1-inch 
eyepiece  and  i-inch  objective.  Where  the  number  of  cell  ele- 
ments exceed  12  or  15  per  microscopic  field,  one-fourth  of  the 
entire  ruled  area  of  the  counter,  equivalent  to  100  of  the  smaller 
squares  of  the  cell,  is  counted.  Where  the  cell  elements  are 
less  abundant,  one-half  of  the  entire  area  (two  to  four  hundred 
squares)  is  examined.  The  average  number  of  cells  per  smallest 
square  is  then  obtained,  which  when  multiplied  by  200,000  gives 
the  number  of  cells  per  cubic  centimeter  in  the  original  milk: 
multiplication  by  four  million  gives  the  number  of  cells  per  cubic 
centimetre  in  the  sediment  examined.  As  the  sediment  repre- 
sents the  cgncentration  of  cells  into  one-twentieth  of  the  orig- 
inal volume  of  milk  taken  (10  c.c.  to  one-half  c.c.)  this  number 
should  be  divided  by  twenty  to  give  the  number  of  cells  per 
cubic  centimetre  in  the  original  milk. 

Expression  of  Results.  All  results  should  be  expressed  in 
number  of  cells  per  cubic  centimetre  of  the  original  milk,  and, 
in  order  to  avoid  fictitious  accuracy  and  yet  to  express  the 
numerical  results  by  a  method  consistent  with  the  precision  of 
the  work,  the  rules  given  below  should  be  followed: 

NUMBERS  OF  CELLS  PER  C.CM. 

From          1,001  to         10,000  recorded  to  the  nearest         100 

10,001  10,000  500 

50,001  100,000  1,000 

100,001  500,000  10,000 

500,001         1,000,000  50,000 

1,000,001       10,000,000  100,000 

Savage,  in  1905,  independently  worked  out  a  volumetric 
method  based  upon  the  same  principle  as  the  Doane-Buckley 


EXPRESSION  OF  RESULTS  177 

method  but  differing  radically  in  technique.  This  was  pub- 
lished in  1906.  9  The  method  of  Savage  is  the  better  one  of 
the  volumetric  methods,  so  full  details  will  be  given:  1  c.cm. 
of  milk  is  placed  in  a  tube  having  a  capacity  of  15  c.cms.  and 
diluted  with  Toisson's  solution  (see  Appendix)  until  the  tube 
is  almost  filled.  The  tube  used  is  of  special  shape  having  the 
lower  end  about  one-quarter  the  diameter  of  the  general  body 
of  the  tube  and  accurately  graduated  at  1  c.cm.  After  well 
mixing  the  fluids,  the  tube  is  centrifugalised  at  1800  revolu- 
tions per  minute  for  ten  minutes.  After  breaking  up  the  cream 
with  a  clean  rod  the  tube  is  whirled  for  a  further  five  minutes. 
The  supernatant  liquid  is  removed  through  a  fine  tube  by 
means  of  a  vacuum  pump  until  just  1  c.cm.  remains.  After 
distributing  the  cells  as  evenly  as  possible  in  the  sediment,  a 
sufficient  quantity  is  placed  in  the  cell  of  a  Thoma-Zeiss  or 
some  other  convenient  form  of  hsemocytometer  and  the  cells 
counted  in  a  number  of  fields  of  vision.  Savage  recommends 
drawing  out  the  microscope  tube  until  an  exact  number  of 
squares  spans  the  field  of  vision  and  gives  the  following  formula 
for  calculating  the  number  of  cells  per  cubic  m.m. 

56,000?/ 
cells  per  cubic  m.m.  of  milk= 


where  y  =  the  average  number  of  leucocytes  per  field  of  vision, 
d  =  the  number  of  squares  which  just  spans  the  diameter.  This 

56  000 
approximation  of  —  jy  -  is  accurate  to  within  0.5  per  cent. 

The  cells  in  the  ruled  squares  can  also  be  counted  and  the  result 
calculated  as  in  ordinary  blood  work,  but  as  these  represent 
but  a  small  proportion  of  the  total  area  of  the  cell,  errors  due 
to  unequal  distribution  of  the  cells  would  be  proportionately 
greater. 

Hewlett,  Villar  and  Revis  add  6  drops  of  formalin  to  60-70 
c.cms.  of  milk  in  order  to  break  down  aggregations  of  cells  and 
to  prevent  the  cells  being  entangled  in  the  cream  layer.  The 


178  CELLS,  DIRT  AND  DEBRIS 

heating  of  the  diluted  milk  tubes  to  70°  in  Savage's  method 
before  centrifugalising  would  possibly  produce  higher  results. 

In  1910  Prescott  and  Breed  10  suggested  the  examination  of 
the  milk  directly  by  means  of  stained  smears.  They  found  the 
results  obtained  by  this  method  to  be  very  much  higher  than 
by  the  Doane-Buckley  method  and  that  they  were  also  more 
consistent.  This  was  due  to  the  varying  number  of  cells 
trapped  by  the  rising  fat  globules.  Breed  afterwards  devel- 
oped the  process  given  on  p.  129  which  is  obviously  as  applicable 
to  cell  examination  as  to  the  enumeration  of  bacteria.  As 
previously  mentioned,  the  accuracy  of  this  method  depends 
up  an  the  even  distribution  of  the  cells  and,  if  this  condition 
does  not  obtain,  a  very  large  number  of  fields  must  be  examined 
in  order  to  obtain  a  fair  average.  With  a  cell  count  over  500,000 
per  c.cm.  the  author  has  obtained  good  results  with  this  method 
but  for  smaller  counts  the  method  of  Savage  is  to  be  preferred 
on  account  of  the  factor  for  the  conversion  of  the  cells  per  field 
to  cells  per  unit  volume  being  so  much  smaller. 

Significance.  Despite  the  numerous  investigations  that 
have  been  made  in  Europe  and  America  during  the  last  seven- 
teen years,  the  significance  to  be  attached  to  presence  of  cells 
in  milk  is  still  surrounded  with  difficulties.  It  has  already 
been  pointed  out  that  a  large  number  of  cells  are  to  be  expected 
in  the  secretion  of  such  an  active  organ  as  the  udder  even  under 
normal  physiological  conditions  and  that  stimulus,  whether 
mechanical  or  pathological,  results  in  an  increase  in  numbers. 
As  might  be  anticipated  under  such  conditions  the  difficulty 
lies  in  establishing  what  might  fairly  be  regarded  as  the  normal 
variation  in  the  number  of  cells.  Savage  found  variations 
ranging  from  50,000  to  1,000,000  cells  per  c.cm.  Russell  and 
Hoffmann  found  counts  as  high  as  1,800,000  in  animals  in  which 
there  was  no  history  of  clinical  disease  while  33  per  cent  of  the 
samples  contained  over  500,000  cells  per  c.cm.  Stone  and 
Sprague,11  using  the  Doane-Buckley  method,  examined  two 
healthy  cows  during  the  whole  milking  period  (1,167  samples) 
with  the  following  results: 


SIGNIFICANCE  179 

Samples.  Cell  Count. 

1 . 2  per  cent under  10,000  per  c.cm. 

7.0  10,000  to  20,000 

61 .0  20,000  to  100,000 

29.0  100,000  to  500,000 

1.8  over  500,000 

Breed  and  Stidger,12  using  the  direct  method,  found  varia- 
tions ranging  from  5000  to  20,000,000  cells  per  c.cm.  in  milk 
whcih  they  regarded  as  normal.  Breed  13  examined  122  cows 
which  averaged  868,000  cells  per  cubic  centimetre;  fifty-nine 
gave  counts  under  500,000  per  cubic  centimetre,  36  between 
500,000  and  1,000,000  per  cubic  centimetre,  and  27  gave 
counts  over  1,000,000  per  cubic  centimetre. 

Hewlett  et  al.2  found  that  a  change  of  feed  influenced 
the  cell  count.  As  regards  physiological  influences,  Savage  14 
found  that  the  previous  number  of  calves  and  the  age  of  the 
cow  had  apparently  little  or  no  effect;  just  after  calving  the 
leucocytes  are  increased,  but  after  this  condition  has  subsided 
the  period  since  parturition  has  no  effect  until  secretion  com- 
mences to  diminish.  The  cells  at  this  period  often  show  very 
abnormal  values  though  not  invariably  so  (Breed).  Regarding 
the  relative  proportion  of  cells  in  the  fore  milk  and  middle  milk 
the  evidence  is  inconclusive,  but  it  is  agreed  that  there  is  an 
increase  in  the  number  discharged  in  the  strippings.  There 
are  marked  daily  variations  in  the  number  of  cells  discharged 
and  equally  large  ones  in  the  product  of  the  four  quarters  of  one 
cow,  for  which  no  adequate  explanation  has  been  offered. 
Pathological  conditions  may  increase  the  cell  content  very 
materially.  Savage  14  obtained  cell  counts  as  high  as  368,000,- 
000  per  cubic  centimetre  in  cases  of  mastitis  and  in  these  con- 
ditions he  also  found  that  the  relative  proportions  of  the  cells 
approximated  to  those  found  in  pus.  The  increased  count  was 
particularly  due  to  polymorphonuclear  leucocytes  which  rep- 
resented 75  to  80  per  cent  of  total  number  of  cells.  Even  after 
the  clinical  evidence  of  mastitis  has  disappeared  the  cell  count 
may  continue  to  be  excessive  for  a  considerable  period.  Some 


180  CELLS,  DIRT  AND  DEBRIS 

workers  have  endeavoured  to  find  a  relation  between  the  cell 
count  and  the  number  of  streptococci  and  other  bacteria  but 
with  no  marked  success.  Milk  stasis  has  been  shown  by  many 
observers  to  have  a  profound  effect  on  the  cell  count  by  mark- 
edly increasing  the  number  of  leucocytes. 

Whilst  it  is  impossible  to  formulate  any  rigid  standard  for 
individual  cows  the  author  believes  that  mixed  milk  con- 
taining over  1,000,000  cells  per  cubic  centimetre  as  determined 
by  the  Savage  or  Breed  methods  should  be  regarded  with  sus- 
picion and  the  supply  at  once  investigated.  An  excessive  cell 
count  is  not  sufficient,  per  se,  to  warrant  condemnation  of  a 
supply,  but  if  other  unsatisfactory  conditions  also  exist,  such  as 
large  numbers  of  streptococci,  the  public  should  be  protected 
by  the  exclusion  of  the  supply  until  the  condition  is  abated. 

The  tentative  working  basis  of  1,000,000  cells  per  cubic 
centimetre  is  not  so  low  as  to  prevent  the  possibility  of  passing 
a  sample  of  mixed  milk  from  a  herd  containing  one  case  of 
garget  but  is  sufficiently  so  to  provide  a  reasonable  safeguard 
without  being  oppressive  on  the  producer.  As  a  routine  method 
of  milk  examination,  the  cell  count  has  little  to  commend  it  in 
the  case  of  herd  milk,  but  in  the  examination  of  individual 
cows  it  is  often  of  great  service. 

Dirt  and  Debris.  During  the  present  century  many 
attempts  have  been  made  to  quantitatively  determine  the 
amount  of  dirt  and  debris  in  milk.  Several  methods  have  been 
used,  but  as  there  is  no  agreement  as  to  what  is  to  be  regarded 
as  dirt  these  have  given  results  which,  although  comparable 
among  themselves,  bear  no  relation  to  each  other. 

The  sediment  from  milk  according  to  Delepine  15  consists  of 

(a)  Cells  derived  from  the  udders. 

(6)  Hairs  and  cells  from  the  milker,  or  cows  or  other  farm  animals. 

(c)  Wool,  cotton  or  other  fibres  from  strainers,  etc. 

(d)  Vegetable  and  mineral  matter  derived  either  from  food,  dung  or 
litter  or  from  dirty  utensils  and  wash  water. 

(e)  Algae,  moulds,  and  bacteria  from  various  sources. 

As  the  cells  and  bacteria  are  separately  determined,  the 


DELEPINE,  BABCOCK,  AND  GERBER       181 

estimation  of  the  sediment  somewhat  overlaps  in  that  direc- 
tion and  its  amount,  "  cseteris  paribus,"  should  bear  some 
relation  to  the  number  of  cells  and  bacteria. 

The  methods  that  have  been  proposed  for  the  estimation  of 
the  sediment  in  milk  may  be  divided  into  two  main  groups. 

(1)  Preparation  of  sediment  by  centrifugalisation. 

(2)  Preparation  of  sediment  by  nitration. 

Group  1.  One  of  the  oldest  methods  of  this  type  is  that  of 
Houston 16  who  added  1  c.c.  of  formalin  to  1  litre  milk  and 
allowed  the  mixture  to  stand  in  a  long  tube  with  a  narrow  lower 
graduated  extremity  closed  by  a  glass  tap.  A  primary  reading 
was  obtained  after  twenty-four  hours  by  making  a  direct  obser- 
vation on  the  scale.  The  sediment  was  then  flushed  out  into  a 
small  graduated  tube  and  the  volume  made  up  to  10  c.cms.  with 
slightly  alkaline  water  (0.1  per  cent  Na2COa).  After  cen- 
trifugalisation for  two  minutes,  a  further  observation  was 
made.  This  was  termed  the  "  secondary  reading."  On 
account  of  the  large  volume  of  milk  required,  this  method  has 
not  been  generally  adopted. 

Delepine,  Babcock,  and  Gerber  all  adopted  methods  in 
which  the  milk  was  centrifugalised  for  a  specified  time  and  the 
volume  of  sediment  read  off  directly  on  the  graduated  lower 
extremity  of  the  tube.  Conn  modified  the  usual  centrifugal 
method  by  washing  the  sediment  with  distilled  water  and, 
finally,  collecting  it  in  tared  filter  papers  which  were  after- 
wards dried  and  weighed.  To  convert  the  dry  weight  to  a 
moist  weight  a  factor  was  necessary  and  this  was  found  to 
average  7.  This  factor  was  somewhat  variable  and  depended 
upon  the  nature  of  the  debris.  Re  vis 17  uses  a  tube  having  a 
capacity  of  approximately  70  c.cms. ;  to  this  is  attached  a  small 
glass  cup  by  means  of  a  ground-glass  joint.  Inside  the  con- 
stricted lower  portion  of  the  larger  tube  a  glass  rod  is  ground  in 
to  form  a  plunger  valve. 

In  the  determination,  the  lower  glass  cap  is  fitted  and  50 
c.cms.  of  milk  placed  in  the  tube  which  is  then  whirled  for  five 
minutes  at  2000  revolutions  per  minute.  After  inserting  the 


182  CELLS,  DIRT  AND  DEBRIS 

rod  valve,  the  lower  tube  is  detached,  the  contents  rejected  and, 
after  reconnecting  with  the  lower  tube,  50  c.cms.  of  distilled 
water  are  added  and  the  valve  withdrawn.  After  stirring  the 
sediment  thoroughly  with  a  platinum  needle,  the  tube  and 
contents  are  given  a  further  five  minutes  in  the  centrifuge.  The 
supernatant  liquid  is  removed  as  before  but  prior  to  the  final 
washing  with  distilled  water,  the  sediment  is  treated  with  1 
c.cm.  of  Eau  de  Javelle  (antiformin  may  be  substituted)  for 
the  purpose  of  dissolving  the  leucocytes  and  epithelial  cells. 
After  the  final  washing,  the  valve  is  inserted,  and  the  lower 
cap  removed  and  dried  in  the  water  oven  with  its  contents. 
From  the  weight  so  obtained  the  tare  of  the  cap  is  deducted 
and  a  correction  made  for  a  blank  determination  on  the  mate- 
rials used.  The  dirt  may  be  used  for  a  microscopical  examina- 
tion. According  to  Re  vis,  the  hypochlorite  has  no  action  on 
dirt  constituents,  but  in  view  of  the  well-known  action  of  chlo- 
rine on  cellulose  this  statement  must  be  accepted  with  reserve. 

Group  2.  The  filtration  methods  included  in  this  group 
are  practically  all  based  on  the  filtration  of  a  given  volume  of 
milk  through  a  disc  of  cotton  wool  followed  by  an  inspection  of 
the  disc  for  visible  dirt. 

Tonney 18  suggested  the  use  of  a  small  disc  of  absorbent 
cotton  in  a  Gooch  crucible  and  operated  with  reduced  pressure 
obtained  from  a  water  pump.  This  is  a  fairly  satisfactory  pro- 
cedure for  laboratory  examinations  but  is  usually  precluded  by 
an  insufficiency  of  sample.  This  principle  of  filtration  for  the 
purpose  of  demonstrating  visible  dirt  has  led  to  the  manufacture 
of  many  commercial  types  of  apparatus  which  have  been  used  in 
dairies  and  creameries,  and  by  milk  inspectors,  with  more  or  less 
success.  The  types  now  on  the  market  are  the  Lorenz  or  Wis- 
consin, Stewart,  and  Gerber,  which  use  gravity  filtration,  and 
the  Lorenz  improved  and  Wizard  which  employ  pressure  or 
suction.  A  detailed  account  of  these  has  been  given  by 
Schroeder19  of  the  Health  Department  of  New  York  City, 
but  as  these  are  of  but  very  limited  utility  in  laboratory  work 
they  will  not  be  discussed  "  in  extenso  "  here. 


BIBLIOGRAPHY  183 

Significance  of  Sediment.  If  no  efforts  were  made  by 
producers  and  dairymen  to  remove  sediment  from  milk,  the 
determination  of  the  dirt  and  debris  would  be  an  invaluable 
guide  to  the  care  exercised  in  the  production  and  handling  of 
milk,  but  in  view  of  the  fact  that  strainers  or  slime  separators 
are  in  almost  universal  use,  the  amount  of  sediment  may  bear 
no  relation  whatever  to  the  general  condition  of  the  milk.  It  has 
been  shown  by  many  sanitarians  that  the  suspended  debris 
represents  only  a  small  proportion  of  the  total  dirt  and  if  this 
solid  debris  is  removed  by  filtration  or  separation  the  general 
physical  appearance  ,of  the  milk  might  be  entirely  fallacious. 
The  use  of  cotton  disc  filters  by  sanitary  inspectors  has  accom- 
plished much  in  the  last  few  years  by  demonstrating  to  vendors 
in  an  incontrovertible  manner  the  dirtiness  of  their  product,  but 
no  real  progress  will  be  affected  thereby  if  the  farmer  increases 
the  efficiency  of  his  strainers  instead  of  preventing  the  access  of 
dirt.  There  is  a  possibility  that  sanitarians  may  defeat  their 
own  objects  by  the  placing  too  much  reliance  on  the  disc  test 
and  failing  to  correlate  it  with  the  bacterial  count  and  other 
tests.  Such  "  prima  facie  "  evidence  of  cleanliness  may  be 
nothing  but  a  specious  fallacy. 

BIBLIOGRAPHY 

1.  Stokes  and  Wegefarth.     Med.  News.     1897,71,45-48.     J.  State  Med., 
5,  439. 

2.  Hewlett,  Villar,  and  Revis.     J.  of  Hyg.     1909,  9,  271-278. 

3.  Scannel.     Amer.  Jour.  Pub.  Health.     1912,  2,  962. 

4.  Ernst.     Milk  Hygiene.     Trans,  by  Mohler  and  Eighorn.     Chicago, 

1914. 

5.  Doane  and  Buckley.     Md.  Agr.  Expt.  Sta.,  Bull.  102,  205-223. 

6.  Russell  and  Hoffmann.     J.  Inf.  Dis.,  Supple.     1907,  3,  63-75. 

7.  Russell  and  Hoffman.     Amer.  Jour.  Pub.  Hyg.     1908,  18,  285-291. 

8.  Amer.  Jour.  Pub.  Hyg.     1910.     20,  315-345. 

9.  Savage.     Jour,  of  Hyg.     1906,  6,  123-138. 

10.  Prescott  and  Breed.     J.  Inf.  Dis.     1911,  7,  632-640. 

11.  Stone  and  Sprague.     Jour.  Med.  Research.     20,  235. 

12.  Breed  and  Stiger.     J.  Inf.  Dis.     1911,  8,  361-385. 

13.  Breed.     New  York  Expt.  Sta.,  Bull.  No.  38.     1914. 


184  CELLS,  DIRT  AND  DEBRIS 

14.  Savage.     Rpt.  of  M.  O.  to  L.  G.  B.     1906-07,  228-236. 

15.  Dele"pine.     Rpt.  to  Manchester  Sanitary  Committee.     1908. 

16.  Houston.     Rpt.  to  London  County  Council.     No.  933.     1905. 

17.  Revis.     Jour.  Roy.  Inst.  Pub.  Health.     1908,  56,  734. 

18.  Tonney.     Amer.  Jour.  Pub.  Health.     1912,  2,  280-281. 

19.  Schroeder.     Amer.  Jour.  Pub.  Health.     1914,  4,  50-64. 


CHAPTER  IX 
PASTEURISED  OR  HEATED  MILK 

IN  addition  to  the  usual  bacteriological  tests  it  is  occa- 
sionally advisable  to  examine  pasteurised  milk  with  a  view  to 
determining  the  nature  of  the  heat  treatment  to  which  it  has 
been  subjected.  Prolonged  heating  at  temperatures  exceeding 
150°  F.  results  in  the  destruction  of  the  enzymes  and  the  loss 
of  albumin  and  soluble  phosphates;  the  fat  globules  may  also 
be  so  altered  that  they  do  not  rise  normally  and  so  affect  what 
is  commercially  known  as  the  "  cream  line." 

The  effect  of  time  and  temperature,  the  two  factors  con- 
trolling the  general  effect,  have  been  admirably  expressed  by 
Dr.  North  of  New  York,  in  a  diagram  which,  with  slight  modi- 
fications to  bring  it  into  harmony  with  the  author's  results,  is 
reproduced  on  page  187. 

For  the  detection  of  overheated  milk,  several  methods  are 
available:  (1)  determination  of  the  cream  line,  (2)  enzyme 
reactions,  and  (3)  estimation  of  the  albumin. 

Cream  Line.  Place  100  c.cms.  of  the  sample  in  a  cream- 
ometer  or  graduated  cylinder  and  observe  the  percentage  of 
cream  obtained  after  standing  for  six  hours  at  60°  F.  If  less 
than  2.5  per  cent  of  cream  rises  for  each  1  per  cent  of  fat  con- 
tained in  the  original  milk,  the  presence  of  heated  milk  must  be 
suspected.  If  less  than  2.5  per  cent  of  cream  is  found  for  each  1 
per  cent  of  fat,  the  sample  may  either  be  milk  pasteurised  at  a 
temperature  exceeding  150°  F.,  or  a  mixture  of  sterilised  and 
fresh  milk. 

Enzymes.  The  effect  of  heat  on  milk  enzymes  has  been 
studied  by  many  workers  and  the  more  important  results  are 
given  in  Table  LXI. 

185 


186  PASTEURISED  OR  HEATED  MILK 

TABLE  LXI 
EFFECT  OF  HEAT  ON  ENZYMES  IN  MILK 


Enzyme. 

Authority. 

WEAKENED 

DESTROYED 

At  Temp. 
0  C. 

in 
Minutes. 

At  Temp. 
0  C. 

In 

Minutes. 

Galactase  

Babcock  and 
Russell 
Von  Freudenreich 
Hippius 

65-70 
65-70 
65 

10 
30 
30 

76-80 
75-80 

Amylase 

Koning 
Hippius 
Race 

68 
75-80 
83 

30 
30 

68 

30 

Lipase 

Gillet 

es 

Lactokinase.  .  . 

Hougardy 

75 

30 

Oxidases 

Marfan 
Hippius 

79 
76 

Peroxidases.  .  . 

Wender 
Schardinger 
Ostertag 
Lythgoe 
Race 
Numerous  others. 

83 
80 
80 
75 
73 
79-80 

30 
30 

70 
68 
75 

30 
30 

Catalase  

Van  Italie 
Wender 

63 
80 

30 

Reductase  .... 

Jensen 
Lythgoe 
Race 

over  70 
70 
71 

30 
30 

65 

68 

30 
30 

Although  these  results  are  slightly  discordant,  they  all  show 
that  thirty  minutes  treatment  at  temperatures  less  than  65°  C. 


ENZYMES 


187 


(149°  F.)  has  no  effect  on  the  enzymes  usually  found  in  fresh 
milk. 

The  tests  most  easily  applied  are  the  hastened  reductase 


180i 


DIAGRAM  No.  IV 


170 


'150 


(140 


Fat,  Sugar, 
Casein,  Salts 


Taste 


Albumin 
Enzymes 


Cream  Line 


130 


10 


20  30  40 

Time  in  Minutes 


Tuberculosis 
Typhoid 

Streptococci 
Diptheria 


reaction  by  means  of  Schardinger's  reagent,  and  the  peroxidase 
reaction    with    benzidine    (page   91).     The  intensity   of  the 


188 


PASTEURISED  OR  HEATED  MILK 


peroxidase  reaction  is  inversely  proportional  to  the  intensity 
of  the  heat  treatment  and  a  similar  indication  is  given  if  more 
than  twenty  to  twenty-five  minutes  are  required  to  discharge 
the  blue  colour  in  the  reductase  test. 

The  results  obtained  by  the  author  on  the  effect  of  heat  on 
the  peroxidase  and  reductase  tests  are  given  in  Tables  LXII  and 
LXIII. 

TABLE  LXII 
EFFECT  OF  HEAT  ON  PEROXIDASE  TEST 


Duration  of 
Heating  in 
Minutes. 

BENZIDINE  REACTION  AFTEK  HEATING  TO 

145°  F. 

150°  F. 

155°  F. 

160°  F. 

165°  F. 

170°  F. 

5 

+ 

+ 

+ 

+ 

+ 

•f 

10 

+ 

-f 

-f- 

-}- 

-}- 

-j- 

15 

+ 

+ 

+ 

+ 

+ 

Faint 

20 
25 

J 

J 

J 

Faint 

Faint 

Very  faint 

30 

+ 

+ 

+ 

Very  faint 

— 

— 

TABLE  LXIII 
EFFECT  OF  HEAT  ON  REDUCTASE  TEST 


Duration  of 


Time  (Minutes)  Required  for  Discharge  of  Colour  after  Heating 
to     (Sample  less  blank). 


145°  F. 

150°  F. 

155°  F. 

160°  F. 

170°  F. 

5 

0 

1 

1 

3 

10 

1 

2 

2 

9 

Over  24  hr. 

15 

2 

3 

3.5 

30 

20 

3 

4 

5 

66 

Over  24  hr. 

25 

3 

4 

6 

204 

30 

4 

6 

7 

Over  24  hr. 

ESTIMATION  OF  ALBUMIN 


189 


If  milk  has  been  treated  with  an  excess  of  hydrogen  peroxide 
or  heated  with  a  smaller  quantity  of  this  substance,  the  perox- 
idases  are  destroyed  and  a  negative  reaction  is  obtained  with 
the  usual  reagents.  Formaldehyde,  in  the  quantities  usually 
employed  for  milk  preservation,  has  no  apparent  effect  on  the 
Schardinger  test. 

Estimation  of  Albumin.  The  estimation  is  most  readily 
performed  in  the  manner  described  on  page  74. 

Rupp  1  obtained  the  following  results  with  heated  milk. 


Milk  Heated  for  Thirty 
Minutes  at 


Percentage  of  Albumin 
Precipitated. 

62.8°  C.  (145°  F.) Nil 

65.6°  C.  (150°  F.) 5.75 

68.3°  C.  (155°  F.) 12.75 

71.1°C.  (160°  F.) 30.87 

The  rennin  coagulation  may  also  be  used  for  the  detection 
of  sterilised  milk  or  milk  heated  at  temperatures  exceeding 
65°  C.  Rupp's  results  (vide  supra)  in  this  connection  are 
given  in  Table  LXIV. 


TABLE  LXIV 

Time  required  for  rennin  coagulation  of  raw  and  heated 
milk.  Milk  200  c.cms. :  rennin  solution  (0.15;  100  c.cms.  water) 
5  c.cms. 


Experi- 
ment. 

Raw  Milk. 

MILK  HEATED  FOR  THIRTY  MINUTES  AT 

55°  C. 
131°  F. 

60°  C. 
140°  F. 

65°  C. 
149°  F. 

70°  C. 
158°  F. 

75°  C. 
167°  F. 

1 
2 

Min.  Sec. 
18  30 
19  08 

19  34 
19  23 

Min.     Sec. 

17      28 
16      56 

Min.     Sec. 

17      10 
16      53 

Min.     Sec. 
17        12 

17      12 

Min.     Sec. 

20      38 
20      25 

Min.     Seo. 

36      30 
37      30 

190  PASTEURISED  OR  HEATED  MILK 

BACILLUS  ABORTUS 

Since  1897  when  Bang  and  Stribald  2  isolated  B.  abortus  as 
the  causative  agent  of  the  infectious  abortion  in  cattle,  con- 
siderable study  has  been  given  to  this  organism  in  various  parts 
of  the  world.  McFadyean  and  Stockman3  corroborated 
Bang's  findings,  but  later  work  has  resulted  in  the  discovery 
of  several  allied  forms  with  the  consequence  that  B.  abortus  is 
now  regarded  as  a  species  and  not  as  a  distinct  biotype. 

During  the  last  decade  several  workers  have  found  B.  abortus 
in  milk  by  the  inoculation  method  and  in  some  instances  as 
many  as  60  per  cent  of  the  samples  gave  positive  results.  The 
lesions  produced  by  these  samples  were  not  usually  sufficient 
to  cause  death. 

Although  the  descriptions  of  B.  abortus  as  given  by  various 
workers  showed  considerable  variations,  it  remained  for  Evans  4 
to  classify  the  various  forms  and  to  indicate  the  relative  fre- 
quency of  certain  varieties  in  normal  udders.  By  plating  milk 
on  agar  containing  10  per  cent  of  bovine  serum,  Evans  isolated 
B.  abortus  from  45  (23.4  per  cent)  of  the  192  samples  exam- 
ined. These  samples  were  obtained  from  5  dairies.  Thirty- 
three  cultures  exhibited  a  marked  lipolytic  action  on  milk  fat 
and  were,  consequently,  designated  as  B.  abortus  variety 
lipolyticus.  Twelve  cultures  (variety  6)  differed  from  the 
pathogenic  varieties  in  their  'ability  to  ferment  the  usual  test 
substances,  and  morphology.  The  reactions  of  the  varieties 
isolated  by  Evans  are  given  in  Table  LXV,  together  with  those 
of  the  typical  pathogenic  varieties  for  comparison. 

B.  abortus  in  young  cultures  shows  the  typical  slender  rod 
form  but  involution  forms  are  often  found  in  older  ones  and 
foetal  exudates  often  contain  coccoid  varieties.  Ordinary 
aniline  dyes  may  be  used  for  staining  purposes,  carbol  fuchsin 
followed  by  1  per  cent  acetic  acid,  dilute  carbol  fuchsin,  and 
Loeffler's  methylene  blue  giving  very  satisfactory  results. 
The  organism  is  decolourised  during  Gram's  method  of  staining. 
For  cultural  preparations  agar  containing  10  per  cent  of  serum 


BACILLUS  ABORTUS  191 

may  be  used  or  an  agar  gelatine  serum  mixture  (4  per  cent 
gelatine,  6  per  cent  agar  and  1  per  cent  serum)  the  serum  in 
which  is  previously  heated  to  60°  C.  for  one  hour  on  4  con- 
secutive days  to  ensure  sterility.     This  latter  medium  is  very 
satisfactory  for  shake  cultures.     In  carbohydrate  media  slightly 
variable  results  are  recorded.    Most  workers  report  that  neutral 
carbohydrate  broths  remain  neutral  or  are  rendered  slightly 
alkaline,  except  for  a  few  cultures  which  produce  slight  acidity 
in  dextrose.     Good  and  Corbett 5  report  that  B.  abortus  variety 
equinus  showed  an  average  of  2  per  cent  of  gas  in  lactose  in  93 
cultures  and  no  gas  in  23.     In  saccharose  58  gave  a  little  less 
than  2  per  cent  of  gas  and  28  were  negative.     Some  cultures 
also  produced  marked  quantities  of  gas  in  xylose,  dextrose, 
arabinose,   dulcite,   sorbite,   mannite,   maltose,   and  raffinose. 
Duplicate  and  triplicate  tests  with  lactose  and  saccharose  gave 
varying  results  but  Good  and  Corbett  are  .convinced  that  the 
gas  produced  is  the  result  of  chemical  action  and  not  adven- 
titious.    From   the  results   of  the   fermentation   tests,   these 
workers  place  the  equinus  variety  in  the  Gaertner  group  of 
organisms.     The  great  difference  in  fermentative  ability  be- 
tween this  variety  and  the  other  members  of  the  B.  abortus 
group  would  appear  to  warrant  a  change  in  the  nomenclature 
of  the  equinus  variety  and  its  removal  from  the  abortus  group. 
In  guinea  pigs,  milk  containing  B.  abortus  often  produces  a 
nodular  condition  of  the  spleen  and  liver,  the  macroscopical 
appearance  having  a  somewhat  superficial  resemblance  to  that 
produced  by  B.  tuberculosis.     In  pregnant  test  animals,  inoc- 
ulation with  cultures  usually  produces  abortion  in  a  few  days 
but  in  some  cases  the  action  is  much  delayed  and  in  others  the 
gestation  period  may  be  quite  normal. 

ACID-PEODUCING   OEGANISMS 

Although  the  organisms  found  in  milk  capable  of  fermenting 
lactose  with  the  production  of  acid  include  such  widely  differ- 
ing groups  as  diplococci,  staphylococci,  streptococci,  and  bacilli, 


192 


PASTEURISED  OR  HEATED  MILK 


TABLE 
COMPARATIVE  CHARACTERISTICS  OF  SEVERAL 


B.  abortus  from  Original 
Descriptions. 

B.  abortus  from   Pathogenic 
Sources. 

Morphology. 

Small  rods,  the  largest 
as  long  as  the  tuber- 
cle bacilli.     (Bang.) 

Slender    rods,    0.8    to 
1.5  microns  in  length. 

Reaction  in  dextrose, 
maltose,  lactose,  raf- 
finose,  mannite,  and 
glycerine  broth. 

Alkaline  broth  is  given 
an     amphoteric     or 
slightly     acid     reac- 
tion    to     Tournesol 
paper.     (Nowak.) 

Neutral  broth  is  ren- 
dered slightly  alka- 
line, except  that  a  few 
cultures  form  a  slight 
acidity  in  dextrose. 

Decomposition  of  ni- 
trogenous com- 
pounds. 

Nitrate,  asparagin  and 
urea  are  commonly 
decomposed.  Gela- 
tine is  not  liquified. 

Action  in  litmus  whole 
milk. 

Rendered  slightly  alka- 
line. 

Growth  in  agar  shake. 

Growth  in  colonies  is 
confined  to  a  zone  of 
from  10  to  15  mm. 
This  zone  lies  about 
5  mm.  under  surface 
of  the  agar.  (Bang.) 

Good  growth  on  sur- 
face. Sometimes  a 
growth  throughout  a 
zone  of  several  mm. 
at  the  top.  Rarely  a 
diaphragm  growth. 

Growth  on  plain  in- 
fusion agar  slope. 

Separate  colonies  re- 
semble rose  coloured 
droplets  reflecting  a 
greenish  tinge. 

Abundant  compact 
growth  chamois  and 
cream  buff  in  col- 
our. 

Growth  in  glycerine 
broth. 

A  poor  growth.  A 
fine  sediment  is 
thrown  down  made 
up  of  whitish  grains. 
(Bang.) 

Good  growth  which 
clouds  the  medium. 

Effect  of  serum  in  the 
agar. 

Growth  is  greatly  fav- 
oured. (Bang.) 

Abundant          growth 
without  serum. 

B.  ABORTUS  GROUP 


193 


LXV 

VARIETIES  OF  BACILLUS  ABORTUS.     (AFTER  EVANS) 


B.  abortus  Lipolyticus. 

B.  abortus  Variety  b. 

B.  abortus  Variety  c. 

Slender  rods,  0.8  to  1.5 
microns  in  length. 

Slender  rods,  0.8  to  1.5 
microns  in  length. 

Slender  rods,  0.8  to 
1.5  microns  in  length. 

No  change. 

Dextrose  and  maltose 
broths  are  rendered 
acid.  No  change  in 
other  broths. 

Slightly  alkaline. 

Nitrate,  and  asparagin 
not  decomposed;  urea 
rarely.  Gelatine  not 
liquefied. 

Nitrate,  asparagin,  and 
urea  usually  decom- 
posed. Gelatine  is 
not  liquified. 

Nitrate,  asparagin  and 
urea  sometimes  de- 
composed. Gelatine 
sometimes  liquified. 

Acid  is  developed  in 
the  cream  layer. 

Slightly  alkaline  in 
most  cases.  No 
change  in  others. 

No  change. 

Colonies  confined  to  a 
thin  layer  a  few  mm. 
beneath  the  surface. 

Similar  to  those  from 
pathogenic  sources. 
Colonies  sometimes 
scattered  throughout 
the  entire  depth  of 
agar. 

Similar  to  the  cultures 
from  pathogenic 
sources. 

A  few  cultures  resem- 
ble those  from  patho- 
genic sources.  The 
growth  scanty  in 
separate  colonies. 

Similar  to  the  cultures 
from  pathogenic 
sources. 

Similar  to  cultures 
from  pathogenic 
sources. 

Scanty  growth  which 
does  not  cloud  the 
medium.  Sediment 
is  made  up  of  little 
granules. 

Abundant  growth. 
The  medium  is  usu- 
ally clouded,  but 
sometimes  the  growth 
is  precipitated,  leav- 
ing a  clear  medium. 

Similar  to  the  growth 
of  variety  b. 

Growth  greatly  fav- 
oured. 

Abundant  growth 
without  serum. 

Abundant  growth 
without  serum. 

194  PASTEURISED  OR  HEATED  MILK 

it  is  sometimes  desirable,  as  in  the  study  of  the  effect  of  heat  or 
chemical  germicides  upon  the  bacterial  flora,  to  determine  the 
relative  proportion  of  this  group  to  the  total  bacteria,  without 
reference  to  the  morphological  characters  of  the  individual 
members. 

This  division  of  the  flora  into  groups  on  an  acid-producing 
basis  is  necessarily  an  empirical  one,  but  it  is  comparatively 
simple  and  has  proved  useful  on  many  occasions. 

The  development  of  this  method  and  its  application  to  the 
examination  of  raw  and  pasteurised  milk  is  largely  due  to 
Ayers  and  Johnson  of  the  United  States  Department  of  Agri- 
culture. In  their  earlier  work  they  grouped  the  flora  into  acid 
forming,  alkali  forming,  inert,  and  peptonising  organisms  ac- 
cording to  their  action  on  litmus  lactose  gelatine.  This  was 
effected  by  plating  out  the  sample  on  this  medium  and  counting 
the  various  groups  after  incubation  for  five  days  at  18°  C.  By 
this  method  it  is  often  difficult  to  distinguish  between  the  feeble 
acid  formers,  the  feeble  alkali  formers,  and  the  inert  group,  but 
fairly  satisfactory  results  have  been  obtained  with  it  in  the 
author's  laboratory  6  and  it  has  the  advantage  of  being  much 
quicker  and  simpler  than  the  later  developments.  The  first 
modification  made  by  Ayers  7  was  an  effort  to  obtain  a  more 
accurate  count  of  the  peptonising  group  by  the  elimination  of 
spoilt  plates  caused  by  the  spread  of  the  gelatine  liquefiers.  A 
neutral  lactose  casein  medium  (see  Appendix)  was  substituted 
for  litmus  lactose  gelatine  and  the  peptonisers  differentiated 
by  flooding  the  surface  of  the  medium,  after  six  days  incuba- 

N 

tion  at  30°  C.,  with  —  lactic  acid.  The  colonies  of  peptonis- 
ing organisms  became  white  owing  to  the  precipitation  of  casein 
by  the  acid.  Ayers,  in  the  same  report  (p.  227)  also  suggested 
the  division  of  the  flora  into  five  groups  according  to  the  action 
on  litmus  milk.  The  colonies  developing  on  lactose  casein  agar 
or  infusion  agar  were  fished  into  litmus  milk  tubes  and  incu- 
bated for  fourteen  days  at  30°  C.  According  to  the  appear- 
ance of  the  milk  after  this  period  the  organisms  were  classified 


ACIDURIC  BACILLI 


195 


as  acid  forming  and  coagulating,  acid  forming,  inert,  alkali 
forming,  and  peptonising.  A  comparison  of  the  milk  tube 
method  arid  the  litmus  lactose  gelatine  plates  was  made  by 
Ayers  and  Johnson  8  who  obtained  the  following  results  as  the 
averages  of  four  samples. 


Acid. 

Alkali  and  Inert. 

Peptonising. 

After  heating  to  140  °F.: 
Milk  tubes      

71  5 

22   8 

5  7 

L.  L.  G.  plates  

43.7 

53  5 

2  8 

After  heating  to  150°F.: 
Milk  tubes 

84  6 

10  5 

4  9 

L.  L.  G.  plates  

41  .2 

57^7 

1.1 

The  milk  tube  method  possesses  the  advantage  of  differen- 
tiating those  organisms  having  feeble  fermentative  ability  and 
also  develops  a  larger  proportion  of  peptonisers.  The  latter 
result  may  be  partially  due  to  the  nature  of  the  nitrogenous 
substance  used  for  the  test  as  it  is  exceedingly  improbable 
that  proteolysis  proceeds  at  the  same  rate  with  all  test  sub- 
stances. 

Aciduric  Bacilli.  Among  the  acid-producing  organisms, 
one  sub-division,  that  of  the  aciduric  or  acidophylic  bacteria, 
is  especially  worthy  of  further  mention  because  it  contains  the 
commercially  important  B.  bulgaricus.  This  organism  has 
achieved  considerable  repute  during  the  last  few  years  as  a 
therapeutic  agent  by  reason  of  its  influence  on  the  flora  of  the 
intestinal  canal  and  it  has,  consequently,  become  necessary 
to  make  bacteriological  examinations  of  the  tablets  used  for 
this  purpose. 

Although  the  aciduric  bacilli  grow  luxuriantly  in  dextrose 
and  lactose  broth  containing  acetic  or  lactic  acid  they  usually 
grow  very  sparingly  or  not  at  all  on  the  usual  laboratory  media. 
They  vary  considerably  in  length  (3  to  7  M)  and  occur  singly  or  in 
chains  or  threads.  They  develop  under  both  aerobic  and  anaer- 
obic conditions  and,  although  typically  Gram  positive,  old  cul- 


196 


PASTEURISED  OR  HEATED  MILK 


tures  may  be  Gram  negative.  Spore  formation  is  never  ob- 
served and  they  ferment  carbohydrates  with  the  production  of 
acid  but  do  not  form  gas.  Milk  coagulation  is  produced  by  some 
members  of  the  group  and  not  by  others. 

For  the  isolation  of  this  group  there  is  no  better  method 
than  that  used  by  Hey  man  in  1898,  viz.,  the  use  of  a  meat  pep- 
tone broth  containing  2  per  cent  dextrose  and  0.3  per  cent 
acetic  acid.  After  incubation  at  37°  C.  for  forty-eight  hours, 
a  portion  of  the  culture  is  seeded  into  another  broth  tube  and 
the  process  repeated  until  only  aciduric  bacilli  remain.  For 
further  isolation  dextrose  agar  containing  1.5  per  cent  agar 
and  2  per  cent  dextrose  without  any  adjustment  of  the  acidity 
may  be  used.  According  to  Rahe  9  the  addition  of  0.2  per  cent 
of  sodium  oleate  as  recommended  by  Salge  10  is  productive  of 
good  results.  By  this  method  Rahe  (vide  supra)  investigated 
a  number  of  the  aciduric  bacteria,  and  divided  them  into  three 
groups  according  to  their  biochemical  properties. 


GROUP. 

A. 

B. 

C. 

Milk  

Clot 

Clot 

No  clot 

Maltose 

Not  fermented 

Fermented 

Fermented 

Group  A,  which  is  the  B.  bulgaricus  group,  is  characterised 
by  a  rapid  clotting  of  milk  and  its  usual  inability  to  ferment 
carbohydrates  other  than  lactose  and  dextrose. 

Group  B  also  clots  milk  but  ferments  maltose,  saccharose, 
and  laevulose  in  addition  to  lactose  and  dextrose,  and  usually 
also  mannite  and  raffinose. 

Group  C  does  not  clot  milk  and  ferments  maltose  even 
more  vigorously  than  group  B.  Saccharose  and  laevulose  are 
fermented  and  usually  raffinose,  but  mannite  is  not  acted  upon. 


FERMENTATION  TEST  197 

THE  FERMENTATION  TEST  IN  MILK  EXAMINATION 

This  test  is  performed  by  incubating  the  sample  in  sterile 
vessels  and  observing  the  chemical  and  physical  changes  that 
take  place. 

The  earliest  experimental  work  in  this  connection  was  prob- 
ably that  of  Walter,  cantonal  chemist  at  Soleure.  This  ob- 
server kept  milk  at  98°  F.,  and  stated  that  "  milk,  if  good,  will 
not  curdle  or  undergo  abnormal  fermentation  in  ten  to  twelve 
hours."  A  special  apparatus  was  devised  for  this  purpose  by 
Schaffer,11  who  recorded  the  amount  of  gas  evolved  in  100°  F. 
from  a  definite  volume  of  milk.  He  found  that  good  milk 
formed  no  gas  and  remained  fluid  after  twelve  hours.  This 
test  was  chiefly  used  in  connection  with  the  suitability  of  milk 
for  cheese  manufacture;  milks  that  produced  "  heaving " 
were  detected  by  this  test. 

The  Wisconsin  curd  test 12  was  also  evolved  for  cheese 
manufacture  and  differs  from  the  Swiss  tests  given  above  in  the 
use  of  rennet  for  the  production  of  a  definite  curd  which  is 
pressed  and  afterwards  set  aside  for  observation. 

The  Gerber  fermentation  test  consists  in  incubating  tubes  of 
milk  at  104°  to  106°  F.  for  six  hours  and  then  observing  the 
odour,  taste,  and  appearance  for  abnormal  qualities.  The 
heating  is  then  continued  for  a  second  six-hour  period  and  any 
abnormal  coagulations,  such  as  gas  holes,  are  then  noted. 
Gerber  stated  that  coagulation  in  less  than  twelve  hours  is 
abnormal,  and  that  milk  that  does  not  curdle  in  twenty-four 
hours  to  forty-eight  hours  is  open  to  suspicion  regarding 
preservatives. 

According  to  Jensen,13  the  milk  is  heated  to  30°  to  35°  C. 
for  eight  to  twelve  hours  and  examined;  replaced  for  a  further 
period  and  again  examined.  After  the  second  period  he  found 
that  the  clean  samples  are  sour  and  curdled  and  form  a  homo- 
geneous coagulum  without  much  separation  of  curd  and  gas 
formation.  Frequently  gas  bubbles  have  split  the  coagulum 
and  considerable  fluid  has  separated.  This  change,  he  states, 


198  PASTEURISED  OR  HEATED  MILK 

does  not  necessarily  signify  that  the  milk  was  particularly  rich 
in  bacteria  of  putrefaction.  If  curdling  is  accompanied  by  an 
offensive  odour  or,  if  the  coagulum  is  peptonised,  the  presence  of 
putrefactive  bacteria  is  inferred.  He  continues,  "  by  boiling 
milk  a  short  time  and  then  incubating,  only  spore  formers 
develop,  and  as  these  are  not  checked  by  the  lactic  bacteria, 
they  increase  rapidly  and  cause  the  milk  to  curdle  by  the  action 
of  ferments.  Pasteurised  milk  does  not  sour,  but  no  precipitate 
conclusions  should  be  drawn  from  the  results  of  this  test." 

Peter,14  Dugelli,15  and  Klein  l6  have  used  thi$  test  for  milk 
examination  and  find  that  it  gives  the  prevailing  types  of  micro- 
organisms with  a  considerable  degree  of  accuracy.  A  combina- 
tion of  the  fermentation  test  with  the  methylene  blue  reduction 
test  has  been  recommended  by  Lohnis  and  Schroeter,17  and  by 
Fred  and  Chappelean.18 

In  1914  the  author  compared  the  results  obtained  by  this 
test  with  the  usual  bacterial  count  on  agar  (forty-eight  hours  at 
blood  heat)  and  the  B.  coli  count  in  rebipelagar.  The  samples 
were  transferred  to  sterile  tubes  plugged  with  absorbent  cotton 
and  incubated  at  37°  C.  (98.5°  F.)  for  20-24  hours.  787  sam- 
ples of  ordinary  raw  milk,  98  samples  of  pasteurised  milk,  and 
69  samples  of  nursery  milk  were  examined  in  this  way  and  the 
results  recorded  according  to  the  classification  of  Dugelli 
(vide  supra).  This  classification,  together  with  the  bacterial 
flora  which  Dugelli  states  is  indicated  by  each  type,  is  as  follows: 


TYPES  OF  CURD 
Type  A 

Liquid.    The  sample  does  not  show  any  marked  change 
except  perhaps  a  slight  deposit  on  the  bottom  of  the  tube. 

1.  Completely  liquid,  sweet  or  sour  taste. 

2.  Somewhat  coagulated  at  the  bottom  or  on  the  walls. 

3.  A  slight  ring  of  curd  under  the  cream,  but  otherwise 
liquid  and  sour. 


TYPES  OF  CURD  199 

4.  Completely  liquid  or  with  a  slight  separation  of  the  solid 
components  of  the  curd.  Taste  strongly  acid  or  bitter  acid. 

Type  B 

Gelatinous  or  Jelly-like.  The  sample  is  more  or  less 
curdled  and  the  casein  is  united  into  a  gelatin-like  mass  without 
any  marked  separation  of  the  curd. 

1.  A  beautiful,  smooth  gelatinous  mass  without  curd  sepa- 
ration and  a  pure  acid  flavour. 

2.  Smooth  but  some  gas  bubbles  and  furrows. 

3.  Generally  smooth,  but  with  curd  separation  and  marked 
by  gas  bubbles  and  furrows. 

4.  Generally  smooth,  with  curd  separation,  but  with  nu- 
merous gas  bubbles  and  furrows. 

Type  C 

Granular.  The  milk  curdles,  but  the  curd,  instead  of  being 
smooth  consists  of  many  small  grains.  Between  the  more  or 
less  fine  curd  grains,  creamy  cheese-like  particles  may  be  found. 

1.  Curd  only  partly  granular  and  partly  gelatin-like  with 
little  cheese  separation. 

2.  Curd  of  fine  granular  structure  and  uniformly  divided  so 
that  the  curd  looks  white. 

3.  Curd  shows  a  marked  separation  with  mostly  large  grains. 

4.  Large  granules  and  complete  coagulation  with  a  creamy 
deposit. 

Type  D 

Cheese  Curd.  The  casein  is  flocculent  or  in  clumps,  and  is 
attached  to  the  sides  of  the  vessel.  The  curd  is  more  or  less 
completely  separated  from  the  whey. 

1.  Casein  is  a  soft,  united  mass.     The  curd  is  greenish  in 
colour  and  slightly  acid. 

2.  Casein  is  a  firm  mass,  curd  green,  and  slightly  acid. 

3.  Casern   pulled   apart   and   divided,    a   greenish   white, 
strongly  acid  curd. 


200  PASTEURISED  OR  HEATED  MILK 

4.  Casein  entirely  separated  and  attached  to  the  sides  of  the 
tube.  A  white  curd,  strongly  acid. 

Type  E 

Gaseous.    The  tube  is  well  marked  with  gas  bubbles. 

1.  Cream  filled  with  bubbles. 

2.  Cream  and  curd  filled  with  bubbles. 

3.  Bubbles  so  numerous  that  the  curd  floats  on  the  whey  and 
forms  a  raised  surface. 

4.  The  gas  development  is  so  pronounced  that  the  curd  is 
forced  upwards  in  the  tube,  often  forcing  out  the  stopper. 

Bacteria  Flora,  as  indicated  by  Fermentation  Test.  (Dugelli.) 

Type  A 

Bacteria  present  in  very  small  numbers.  Cocci  predominate 
with  few  lactic  acid,  coli  and  serogenes  organisms. 

Type  B 

Lactic  acid  in  great  numbers,  few  if  any  coli  and  aerogenes 
organisms,  some  cocci  and  fluorescent  bacteria.  Gas  formation 
indicates  the  presence  of  coli,  aerogenes,  or  butyric  organisms. 

TypeC 

Lactic,  coli,  and  aerogenes  bacteria  predominate  with  many 
cocci. 

Type  D 
Lactic  acid  mixed  with  coli  and  aerogenes  organisms. 

Type  E 

Coli  and  aerogenes  organisms  abound  if  much  gas  is 
formed;  also  lactic  bacteria,  cocci  and  B.  vulgatus. 


TYPES  OF  CURD  201 

The  author's  results  showed  that  the  type  of  fermentation 
was  determined  by  a  combination  of  factors  which  varied  in 
different  samples.  The  chief  factors  were  the  total  and  relative 
numbers  of  the  various  groups  of  organisms  which  constituted 
the  bacterial  flora. 

When  the  total  bacteria  were  very  low  the  fermentation  was 
usually  of  the  A  type,  i.e.,  very  little  visible  alteration  occurred 
in  the  physical  appearance  of  the  sample,  and  a  smooth  acid 
flavour  was  produced.  The  acid  producers  were  so  few  in  num- 
bers as  to  be  unable  to  produce,  under  the  incubator  conditions, 
sufficient  acid  to  coagulate  the  caseinogen.  This  is  the  dis- 
tinguishing feature  of  type  A.  In  types  B,  C,  and  D,  there  was 
a  distinct  coagulation,  but  the  character  varied  in  each  group 
according  to  the  organisms  associated  with  the  acid  producers. 
The  acid  producers  in  each  case  produced  their  effect,  and  if  the 
ratio  of  acid  formers  to  gas  formers  were  large,  little  or  no  evi- 
dence of  gas  formation  was  observed.  As  this  ratio  decreased 
furrows  became  evident  and  numerous  gas  bubbles  were  found 
enclosed  in  the  curd,  whilst  in  extreme  cases  the  gas  formation 
was  so  marked  as  to  force  the  cream  layer  to  the  top  of  the  tube. 
As  any  gas  formed  previous  to  the  production  of  a  firm  curd 
would  be  lost  without  leaving  any  evidence,  it  follows  that  any 
gas  observed  must  have  been  produced  after  coagulation  and  in 
a  medium  of  increased  acidity.  To  effect  this  the  proportion  of 
colon  organisms  must  be  considerable,  as,  otherwise,  their 
development  would  be  retarded  by  the  metabolic  products  of 
the  acid  group.  Very  many  samples,  however,  were  observed 
to  produce  gas  bubbles  in  the  fermentation  test,  and  yet  con- 
tained originally  less  than  one  B.  coli  per  cubic  centimetre. 
In  these  cases  either  the  small  numbers  of  the  B.  coli  must  have 
increased  very  rapidly  in  proportion  to  the  acid  formers  or  be 
of  an  acid  resisting  type.  At  ordinary  temperatures  (50°  to 
60°  F.),  the  colon  content  usually  continued  to  increase  until 
about  0.7  per  cent  of  acidity,  calculated  as  lactic  acid,  was 
produced.  .. 

The  results  also  showed  that  the  same  type  of  fermentation 


202  PASTEURISED  OR  HEATED  MILK 

was  produced  by  very  widely  differing  B.  coli  contents,  and  it 
was,  therefore,  impossible  to  form  a  definite  opinion  regarding 
the  B.  coli  content  from  the  appearance  of  the  fermentation 
test.  The  A  type  was  almost  invariably  produced  by  milk 
low  in  B.  coli,  whilst  D5  pointed  to  excessive  contamination 
with  this  group,  but  with  regard  to  the  intermediate  types, 
which  the  majority  of  market  milks  produce,  no  definite  con- 
clusions could  be  deduced.  The  same  remarks  apply  regarding 
the  relation  of  the  total  bacterial  count  to  the  type  of  fermenta- 
tion, and,  under  these  circumstances,  it  is  difficult  to  attach 
much  value  to  this  test.  Some  observers  have  a  high  opinion 
of  this  test,  because  it  is  supposed  to  yield  evidence  as  to  bac- 
terial flora  and  thus  enable  deductions  to  be  made  as  to  the 
conditions  under  which  the  milk  was  produced  and  its  subse- 
quent treatment,  but  the  author's  results  do  not  substantiate 
this  claim. 

The  conditions  of  the  test,  viz.,  incubation,  at  blood  heat, 
are  artificial,  as  milk  is  never,  under  ordinary  circumstances, 
kept  at  this  temperature,  and  it  is  not  logically  sound  to  assume 
that  the  biological  and  chemical  changes  are  the  same  at  dif- 
ferent temperatures  as  a  change  of  temperature  always  favours 
the  growth  of  one  or  more  groups  in  preference  to  others. 

COLLECTION  OF  SAMPLES 

All  milk  sold  in  bulk  must  be  thoroughly  mixed  before 
samples  are  taken  and  every  endeavour  should  be  made  to 
obtain  milk  in  the  same  manner  in  which  the  vendor  supplies 
the  same  to  the  consumer.  The  Committee  of  the  American 
Public  Health  Association,  appointed  for  the  standardisation 
of  bacteriological  examination  of  milk,  have  recommended  that 
bacteriological  samples  should  be  obtained  from  bulk  milk  by 
means  of  sterile  pipettes,  but  this  method  samples  milk  which 
is  in  the  possession  of  the  vendor  and  ignores  possible  contami- 
nation in  the  vessel  used  for  the  transfer  of  such  milk  to  the 
consumer.  The  author  has  observed  numerous  instances  in 


COLLECTION  OF  SAMPLES  203 

which  this  vessel  has  had  very  appreciable  effects  upon  the  bac- 
terial count  and  the  number  of  coliform  bacteria.  For  the  col- 
lection of  combined  chemical  and  bacteriological  samples  the 
author  has  used  for  several  years  rectangular,  narrow-necked, 
six-ounce  glass-stoppered  bottles,  16  of  which  can  be  placed  in  a 
tray,  10  by  6|  inches.  This  tray  is  surrounded  with  ice  and 
water,  and  the  whole  contained  in  a  water-tight  galvanised-iron 
box  14J  by  10J  by  7  inches.  In  cold  climates  the  cooling 
mixture  can  be  dispensed  with  in  winter  and  when  there  is 
any  possibility  of  the  milk  freezing,  wide-mouthed  bottles 
should  be  used  to  prevent  freezing  of  the  sample  and  so  blocking 
the  neck  of  the  bottle  during  the  transfer  of  the  sample.  All 
milk  retailed  in  bottle  should  be  delivered  to  the  laboratory 
in  the  original  container  unopened  as  the  only  other  method  of 
satisfactorily  sampling  such  milk  is  to  transfer  the  sample  to  a 
sterile  bottle  and  then  back  to  the  original  container,  this  being 
repeated  several  times.-  The  sterile  bottle  necessary  for  the 
success  of  this  method  cannot  usually  be  obtained  so  that  this 
system  should  not  be  encouraged. 

All  samples  should  be  labelled  in  such  a  way  that  there  can 
be  no  possibility  of  doubt  as  to  the  identity  of  each  sample 
and  a  complete  record  of  the  sampling  data  made  immediately 
after  the  sample  is  taken.  This  should  include  name  of  vendor, 
date,  time  and-  place,  temperature,  character  of  container 
and  name  of  collector.  The  temperature  of  milk  in  bulk  is 
observed  immediately  after  the  sample  has  been  taken  whilst 
that  of  bottled  milk  should  be  obtained  from  a  second  bottle. 
A  quickly  reacting  Fahrenheit  thermometer  is  suitable  for  this 
purpose. 

If  the  object  of  the  examination  of  samples  is  to  obtain 
figures  representative  of  the  total  milk  supply  and  from  which 
averages  can  be  calculated  which  are  strictly  comparative  from 
month  to  month  or  from  year  to  year,  the  collection  of  samples 
must  be  carried  out  as  scientifically  as  possible  and  not  in  the 
usual  haphazard  fashion.  The  output  of  each  vendor  should 
be  estimated  and  the  number  of  samples  varied  in  proportion 


204 


PASTEURISED  OR  HEATED  MILK 


m 
o 

o 

o 

o 
g 

S 

PS 

^2 

XI  ^ 

^  3 

gs 

PQ  co 


,-3; 


co  o'.-H£;dcocor^odcOf-Hco     co 


10  co  Tj<  o  •*  10  eo  i»  co  i>  t^-  o 

sssssssssss  s 


O5O5CDO>p<N^HO5Tjt^HO(N 
CqiQ»HC<I^COOOiOt^»OcNO 


<  00  i-t  ^  CO  M  00  M  CD  t»  00 


OS  O  O5  <N  O  CC  O  •*  00  CO  t^  00 


8o§ 
§"§ 


2    § 


M 

1 1 


l^  00  OS  OS  •<*<  O  (N  -<t  •*  CC  C  O       C 


O»  OO  OC  «O  00  rf<  CD  O5  CO  O  l>  «3        i-< 
CO  Tj*  CO  •<*  r}«N  CO  rt  n  ^H  (N  (N       CC 


00  CO  f-H  iO  CO  .-H  CO  <N  O5  CO  t-i  00       O 

t^iCCDTf(T}<i-HC^»C(NOOT}<N       Cl 
CO  rj<  CO  CO  ^  (N  CO  rH  ^H        (N  <N        <N 


i-H  CM  N  (N  CO  CO  CO  Tj<  CO  ^H  CO  CO 


I 

Q^faS<S^^<JccO     •< 


CO 

o 


>-i  00  <N  CO  •*  rt<  00  O        CO 

o 


Is 


it 

S  s^ 


ii 


«5s 


O5  •*  »C  CO  OS  (N  .-H  00  00  .-I  O5  CN   Tj< 

^HCOt^OOGO^-i^-iOOiO^CNCo'   CO 
O  Oi  O5  O5  O5  O  O5  t»  t^  t>-  00  00   00 


00  CO  CO  O  t*  CO  (N  O  •<*<  b-  t~-  00       "5 


C3O3OCCCO*— 1 1>-  O*l  C^J  00  CO  CO   Cl 
!>•  X  O  35  O5  OO  t>-  CO  iO  CC  1C  CD   b- 


I-H  co  •*  10  TJ*  cc  oo  «-•  co  0  1-1   c: 


t^  O  O5  CS1  1^  <N  CO  I-H  CO  I-H  CN  CO   O 

t-IcOt>-*iCCOfO"CXiO'«t^-I   l« 
CD  t^  I>  00  00  CO  IO  CC  <N  1-1  CO  •*   «C 


»O  00  CO  **  CO  1C  t~-  CD  O  O  fH  >-H        CC 

1C  CC  O  CM  ^  CN  1C  M  O  >-H  Tj(  OJ        CC 
CC  Tfi  CC  <N  CC  CO  IN  ^H  01  ,-H  ^  ^H        (N 


<N  (N  CD  00  ^  t^  O5  IO  CO  (N  ^-« 


>oo 

l%% 


^HCN         O5 

SOO-^^HO       CM 
COCOCDT*<       TJ. 


ooTjTcc  co'oo'cD'o^^of  otTo'cT     «b" 

^H  ^  ^,  ,_i  ^  ?J  (M  CO  CO  CO  (N  (N        (N 


i 


RECORDING  RESULTS 


205 


TABLE  LXVII 

PASTEURISED  MILK  YEAR  ENDING  OCTOBER  31,  1915. 

BACTERIOLOGICAL 


OTTAWA 


Month. 

VARIATION  IN  BACTERIAL  COUNT.  PERCENTAGE  OF  SAMPLES  CONTAINING 

Under 
10,000 

10,001 
to 
50,000 

Under 
50,000 

50,001 
to 
100,000 

Under 
100,000 

100,001 
to 
500,000 

Under 
500,000 

November.  .  . 
December.  .  . 
January  
February.  .  .  . 
March  
April  
May 

41.2 
31.2 
53.8 
33.3 
43.8 
25.0 
81.8 
57.9 
57.1 
50.0 
31.6 
33.3 

45.0 

47.0 
62.5 
46.2 
66.7 
56.2 
75.0 
18.2 
21.1 
38.1 
50.0 
63.2 
60.1 

50.4 

88.2 
93.7 
100.0 
100.0 
IGO.O 
100.0 
100.0 
79.0 
95.2 
100.0 
94.8 
93.4 

95.4 

5.9 
6.3 
Nil 
Nil 
Nil 
Nil 
Nil 
21.0 
Nil 
Nil 
5.2 
6.6 

3.7 

94.1 
100.0 

5.9 
Nil 
Nil 
Nil 
Nil 
Nil 
Nil 
Nil 
4.8 
Nil 
Nil 
Nil 

0.9 

100.00 

100.0 
100.0 

!!!  :;; 

June  
July  

95.2 

September.  .  . 
October 

100.0 
100.0 

99.1 

Average  

Month 

Mean 
Bacterial 
Count 
per  c.cm. 

Mean 
B.  coli 
per 
c.cm. 

VARIATION  IN  B.  COLI  PER  C.CM.  PERCENTAGE 
OP  SAMPLES 

Under 
10 

11  to 
50 

Under 
50 

51  to 
100 

Under 
100 

Over 
100 

November.  .  . 
December.  .  . 
January  
February.  .  .  . 
March  
April 

25,000 
19,000 
16,200 
18,000 
14,400 
17,000 
13,000 
22,600 
28,000 
11,000 
33,000 
18,700 

19,600 

3 
15 
9 
3 
35 
6 
7 
55 
141 
46 
896 
6 

102 

88.3 
56.1 
76.9 
93.4 
75.0 
91.7 
86.3 
42.1 
40.0 
50.0 
47.3 
86.7 

69.5 

11.7 
31.2 
15.4 
6.6 
6.2 
8.3 
13.7 
42.1 
20.0 
25.0 
31.6 
13.3 

18.8 

100.0 
87.3 
92.3 
100.0 
81.2 
100.0 
100.0 
84.2 
60.0 
75.0 
78.9 
100.0 

88.3 

Nil 
12.7 
7.7 
Nil 
Nil 
Nil 
Nil 
15.8 
10.0 
12.5 
10.4 
Nil 

5.7 

166!  6 

100.0 

Nil 
Nil 
Nil 
Nil 
18.0 
Nil 
Nil 
Nil 
10.0 
12.5 
10.7 
Nil 

6.0 

81.2 

166!  6 

90.0 
87.5 
89.3 

94.0 

May  
June 

July  

August 

September  .  . 
October.  .  .  . 

Average  

CHEMICAL 


Month. 

SAMPLES  BELOW  STANDARD. 

GENUINE  MILKS. 

Number 
of 
Samples. 

Percentage  of  Samples. 

Average  Composition. 

Deficient 
in  Fat. 
Solids. 

Deficient 
Total 
Solids 

Below  8.  5 
Per  Cent 
Not-fat. 
Solids. 

Fat. 

Total 
Solids. 

Solids 
Not-fat. 

November 

4.03 
3.85 
3.79 
3.73 
3.62 
3.65 
3.70 
3.84 
3.77 
3.86 
3.97 
3.93 

3.81 

13.02 
12.81 
12.70 
12.63 
12.59 
12.46 
12.67 
12.73 
12.52 
12.57 
12.74 
12.97 

12.72 

8.99 
8.96 
8.99 
8.90 
8.97 
8.81 
8.97 
8.89 
8.75 
8.71 
8.77 
9.04 

8.91 

17 
16 
13 
15 
16 
12 
22 
19 
21 
16 
19 
15 
Total 
201 

December 

February 

March 

April     . 

May 

June  

July 

6.4 

Sept 

October 

05 

Average  

206  PASTEURISED  OR  HEATED  MILK 

to  the  output.  When  various  grades  of  milk  are  offered  for 
sale,  the  results  should  be  separately  recorded.  The  interval 
between  sampling  and  examination  should  be  as  short  as  pos- 
sible although  no  appreciable  alteration  occurs  even  in  twenty- 
four  hours  if  the  samples  are  kept  between  32°  and  40°  F. 

Recording  Results.  The  ordinary  method  of  recording 
results  by  expressing  the  average  total  bacterial  count  or  the 
average  number  of  bacteria  of  some  particular  group  of  organ- 
isms, may  give  a  result  which  does  not  represent  the  quality  of 
the  supply  if  the  variations  from  the  mean  are  large,  or  if  the 
number  of  variants  is  comparatively  small.  The  median 
would  be  more  representative  of  the  actual  quality  than  the 
mean  but  a  better  plan  is  to  express  variations  in  the  counts  in 
the  manner  set  forth  in  Tables  LXVI  and  LXVII.  The  size  of 
the  groups  in  the  scheme  is  quite  arbitrary,  but  where  milk  is 
graded  they  should  agree  with  the  limits  permitted  in  each  par- 
ticular grade. 

BIBLIOGRAPHY 

1.  Rupp.     Bull.  166,  U.  S.  A.  Dept.  of  Agr. 

2.  Bang  and  Stribald.     Zeit.  f.  Tiermidicin.     1897,  1,  241-278. 

3.  McFadyean  and  Stockman.     Rpt.  of  departmental  committee  to  the 

Board  of  Agr.,  Appendix  to  Part  1.     London,  1909. 

4.  Evans.     Jour.  Inf.  Dis.     1916,  18,  437-477. 

5.  Good  and  Corbett.     Jour.  Inf.  Dis.     1916,  18,  586-596. 

6.  Race.     Can.  Jour.  Pub.  Health.     1915,  6,  490. 

7.  Ayers.     28th  Rpt.  Bureau  Animal  Ind.,  U.  S.  A.     228. 

8.  Ayers  and  Johnson.     Bull.  161,  Bureau  Animal  Ind.,  U.S.A. 

9.  Rahe.     Jour.  Inf.  Dis.     1914,  15,  143. 

10.  Salge.     Jahrb.  f.  Kinderh.     1904,  59,  309. 

11.  Schaffer.     Landw.  Jahrbuch  der  Schweiz,  7,  72. 

12.  Wisconsin  Expt.  Stat.  Annual  Rpts.     1895  and  1898. 

13.  Milk  Hygiene  by  Jensen.  Trans,  by  Mohler  and  Eichhorn.     Chicago, 

1914. 

14.  Peter.     Jahresb.  d.  Molkereischule  Rutti.     1905-1906.  210. 

15.  Dugelli.     Centralbl.  Bakt.,  11  Abt.,  Bd.,  18,  pp.  37,  224,  439. 

16.  Klein.     Amer.  Vet.  Review.     Oct.,  1912,  25. 

17.  Lohnis  and  Schroeter,  Centralbl.  f.  Bakt.,  II,  Abt.,  Bd.,  32.     1912,  181. 

18.  Fred  and  Chappelean.     Virginia  Agr.  Expt.  Stat.,  1911-1912,  233. 


APPENDIX 


Rebipelagar  or  Neutral  Red  Bile  Salt  Agar: 

Agar 20  grams 

Peptone 20  grams 

Bile  salt  commercial 5  grams 

Water 1CGO  c.cms. 

Heat  the  ingredients  in  a  double  pan  or  autoclave  until 
completely  dissolved;  titrate  with  alkali  and  adjust  the  reac- 
tion to  +1.0  per  cent  to  phenol phthalein.  Cool  to  45°  C., 
coagulate  with  egg  albumen  (5  grams  dissolved  in  water), 
heat  to  boiling,  adjust  the  weight  and  filter.  Tube  in  con- 
venient quantities,  after  adding  5  grams  of  lactose  and  5  c.cms. 
of  a  1.0  per  cent  solution  of  neutral  red. 

Aesculin  Bile  Salt.  (Harrison  and  Vanderleck,  Trans. 
Roy.  Soc.  of  Canada,  1909,  Sec.  IV,  147.) 

Dissolve  in  water  1.0  per  cent  of  Witte's  peptone,  0.25  per 
cent  of  bile  salt,  and  1.5  to  2.0  per  cent  of  agar.  Neutralise 
with  alkali,  coagulate  with  egg  albumen  and  filter.  Add  0.2 
per  cent  of  citrate  of  iron  and  0.1  per  cent  of  sesculin.  This 
amount  of  citrate  of  iron  should  give  a  final  acidity  of  +0.7  per 
cent  and  produces  a  slight  fluorescence  in  the  medium. 

Toissons's  Solution: 

Methyl  violet 0 . 025  gram 

Sodium  chloride 1.0  gram 

Sodium  sulphate 8.0  grams 

Glycerine 30  c.cms. 

Distilled  water 160  c.cms. 

The  solution  should  be  freshly  filtered. 

207 


208  APPENDIX 

Ponder's  Stain.     Kinyoun's  modification. 

Toluidine  blue 0.1  gram 

Azure  1 0.01  gram 

Methylene  blue 0.01  gram 

Glacial  acetic  acid 1.0  c.cm. 

95  per  cent  alcohol 5.0  c.cms. 

Distilled  water 120  c.cms. 

The  films  should  be  stained  for  two  minutes  or  more. 

Dorset* s  Egg  Medium.  Take  12  fresh  eggs,  wash  the  shells 
with  water  and  then  with  undiluted  formalin;  allow  to  dry. 
Break  the  eggs  into  a  graduated  cylinder  and  note  the  total 
volume.  Add  one  part  of  sterile  saline  solution  (0.85  per  cent 
sodium  chloride)  to  three  parts  of  the  mixed  eggs.  Pour  into  a 
sterile  beaker  or  basin  and  whip  with  an  egg  whisk;  filter 
through  cheese  cloth  or  muslin  into  a  sterile  flask  and  tube 
10  c.cms.  in  the  usual  way.  Inspissate  at  75°  C.  for  one  hour  in 
a  sloping  position  and  then  add  0.5  c.cm.  of  sterile  glycerine 
broth  (physiological  saline  containing  6.0  per  cent  of  glycerine) 
to  each  tube  to  prevent  drying.  Incubate  at  37°  C.  for  forty- 
eight  hours  and  reject  all  contaminated  tubes.  Eyre  recom- 
mends adding  sufficient  alcoholic  basic  fuchsin  to  produce  a 
distinct  colouration  before  the  medium  is  tubed. 

Casein  agar.  To  300  c.cms.  of  distilled  water  add  10  grams 
of  casein  (C.  P.  Hammersten)  and  7  c.cms.  of  N.  NaOH.  Heat 
to  boiling  for  several  hours  until  thoroughly  dissolved.  Adjust 
the  weight  and  bring  the  reaction  to  0.2  per  cent  acid.  The 
agar  solution  is  prepared  by  dissolving  10  grams  of  agar  in  500 
c.cms.  of  water.  Both  solutions  are  filtered,  mixed,  tubed, 
and  sterilised  under  pressure.  The  final  reaction  should  be 
+0.1  per  cent  and,  if  the  acidity  is  higher  than  this,  a  portion 
of  the  casein  will  be  precipitated  during  sterilisation. 


APPENDIX 


209 


O 

- 


&&i 


ooccfOeorococccQT^T^T^Tt<Tt<Tt(Tj<>oiciO»o»oiO'CcDcD^>co«ocDt>t^t^ 

COWCCCOCOCCCOCCCOCCcCOOCCCOCCCOCCCOCOCOCOCCCCCOCOMCCMCOfOCC 


^^^^^^^(^(^ 
COCOCOCOCOCOCOCOCO 


COCOWfOfOlXCO 


COCOCOCOeOOCi^TtlT^Tfr^-^TtHiOiO 
COCOWCOl^COl^CCCOCCCOCOMWfC 


cccoeowcocoMWcoccMfoccoo 


C5O5Oi0505OOOOOOOOO--i'-i'-<'HT-l^H^H(N(N(N(N(N(N(NCOCO 


JOOOOO--I 


ooooxooososoiOiaioiasosoiOioooooooO' 


>O>Oi<3>OJgr.gO5OS 


<N(NC<l(NC^C^<NC^C^C^<NC<lC^Cv|C<lC<I(NC<)C<l(NIN 


CS1C^(N(NIN01(NC<JWC<J<NC^(N 


(N<N! 


iN(N<N<N(NC<l(N<N<N(N(N(N 


TjtrfrJtiOiOiOiO'OiO'O'O 


Ttl  Tj<  Tj<  Tj<  Tt< 


210 


APPENDIX 


TABLE 

FOR  CALCULATION  OF  TOTAL  SOLIDS 
ACCORDING  TO  BABCOCK. 

LACTOMETER  READING 


Fat. 

26.0 

26.5 

27.0 

27.5 

28.0 

28.5 

29.0 

29.5 

30.0 

30.5 

31.0 

0.0 
0.1 

0  ?, 

6.50 
6.62 
6  74 

6.62 
6.74 
6.86 

6.75 
6.87 
6  99 

6.87 
6.99 
7  11 

7.00 
7.12 
7  24 

7.12 
7.24 
7  36 

7.25 
7.37 
7  49 

7.37 
7.49 
7  61 

7.50 
7.62 

7  74 

7.62 
7.74 
7  86 

7.75 
7.87 
7  99 

0.3 
0  4 

6.86 
6  98 

6.98 
7.10 

7.11 
7  23 

7.23 
7  35 

7.36 

7  48 

7.48 
7  60 

7.61 
7  73 

7.73 

7  85 

7.86 
7  98 

7.98 
8  10 

8.11 
8  23 

0.5 

7.10 

7.22 

7.35 

7.47 

7.60 

7.72 

7.85 

7.97 

8.10 

8.22 

8.35 

0.6 

7.22 

7.34 

7.47 

7.59 

7.72 

7.84 

7.97 

8.09 

8.22 

8.34 

8.47 

0.7 

7.34 

7.46 

7.59 

7.71 

7.84 

7.96 

8.09 

8.21 

8.34 

8.46 

8.59 

0.8 

7.46 

7.58 

7.71 

7.83 

7.96 

8.08 

8.21 

8.33 

8.46 

8.58 

8.71 

0.9 

7.58 

7.70 

7.83 

7.95 

8.08 

8.20 

8.33 

8.45 

8.58 

8.70 

8.83 

.0 

7.70 

7.82 

7.95 

8.07 

8.20 

8.32 

8.45 

8.57 

8.70 

8.82 

8.95 

.1 

7.82 

7.94 

8.07 

8.19 

8.32 

8.44 

8.57 

8.69 

8.82 

8.94 

9.07 

.2 

7.94 

8.06 

8.19 

8.31 

8.44 

8.56 

8.69 

8.81 

8.94 

9.06 

9.19 

.3 

8.06 

8.18 

8.31 

8.43 

8.56 

8.68 

8.81 

8.93 

9.06 

9.18 

9.31 

.4 

8.18 

8.30 

8.43 

8.55 

8.68 

8.80 

8.93 

9.05 

9.18 

9.30 

9.43 

.5 

8.30 

8.42 

8.55 

8.67 

8.80 

8.92 

9.05 

9.17 

9.30 

9.42 

9.55 

.6 

8.42 

8.54 

8.67 

8.79 

8.92 

9.04 

9.17 

9.29 

9.42 

9.54 

9.67 

.7 

8.54 

8.66 

8.79 

8.91 

9.04 

9.16 

9.29 

9.41 

9.54 

9.66 

9.79 

.8 

8.66 

8.78 

8.91 

9.03 

9.16 

9.28 

9.41 

9.53 

9.66 

9.78 

9.91 

.9 

8.78 

8.90 

9.03 

9.15 

9.28 

9.40 

9.53 

9.65 

9.78 

9.90 

10.03 

2.0 

8.90 

9.02 

9.15 

9.27 

9.40 

9.52 

9.65 

9.77 

9.90 

10.02 

10.15 

2.1 

9.02 

9.14 

9.27 

9.39 

9.52 

9.64 

9.77 

9.89 

10.02 

10.14 

10.27 

2.2 

9.14 

9.26 

9.39 

9.51 

9.64 

9.76 

9.89 

10.01 

10.14 

10.26 

10.39 

2.3 

9.26 

9.38 

9.51 

9.63 

9.76 

9.88 

10.01 

10.13 

10.26 

10.38 

10.51 

2.4 

9.38 

9.50 

9.63 

9.75 

9.88 

10.00 

10.13 

10.25 

10.38 

10.50 

10.63 

2.5 

9.50 

9.62 

9.75 

9.87 

10.00 

10.12 

10.25 

10.37 

10.50 

10.62 

10.75 

2.6 

9.62 

9.74 

9.87 

9.99 

10.12 

10  24 

10.37 

10.49 

10.62 

10.74 

10.87 

2.7 

9.74 

9.86 

9.99 

10.11 

10.24 

10.36 

10.49 

10.61 

10.74 

10.86 

10.99 

2.8 

9.86 

9.98 

10.11 

10.23 

10.36 

10.48 

10.61 

10.73 

10.86 

10.98 

11.11 

2.9 

9.98 

10.10 

10.23 

10.35 

10.48 

10.60 

10.73 

10.85 

10.98 

11.10 

11.23 

30 

10.10 

10.22 

10.35 

10.47 

10.60 

10.72 

10.85 

10.97 

11.10 

11.23 

11.36 

3.1 

10.22 

10.34 

10.47 

10.59 

10.72 

10.84 

10.97 

11.09 

11.22 

11.35 

11.48 

3.2 

10.34 

10.46 

10.59 

10.71 

10.84 

10.96 

11.09 

11.21 

11.34 

11.47 

11.60 

3.3 

10.46 

10.58 

10.71 

10.83 

10.96 

11.09 

11.21 

11.34 

11.46 

11.59 

11.72 

3.4 

10.58 

10.70 

10.83 

10.96 

11.09 

11.21 

11.34 

11.46 

11.58 

11.71 

11.84 

3.5' 

10.70 

10.82 

10.95 

11.09 

11.21 

11.33 

11.46 

11.58 

11.70 

11.83 

11.96 

3.6 

10.82 

10.95 

11.08 

11.20 

11.33 

11.45 

11.58 

11.70 

11.82 

11.95 

12.08 

3.7 

10.94 

11.07 

11.20 

11.32 

11.45 

11.57 

11.70 

11.82 

11.94 

12.07 

12.20 

3.8 

11.06 

11.19 

11.32 

11.44 

11.57 

11.69 

11.82 

11.94 

12.06 

12.19 

12.32 

3.9 

11.18 

11.31 

11.44 

11.56 

11.69 

11.81 

11.94 

12/06 

12.18 

12.31 

12.44 

4.0 

11.30 

11.43 

11.56 

11.68 

11.81 

11.93 

12.06 

12.18 

12.31 

13.43 

12.56 

4.1 

11.42 

11.55 

11.68 

11.80 

11.93 

12.05 

12.18 

12.30 

12.43 

12.55 

12.68 

4.2 

11.54 

11.67 

11.80 

11.92 

12.05 

12.17 

12.30 

12.42 

12.55 

12.67 

12.80 

4.3 

11.66 

11.79 

11.92 

12.04 

12.17 

12.29 

12.42 

12.54 

12.67 

12.79 

12.92 

4.4 

11.78 

11.91 

12.04 

12.16 

12.29 

12.41 

12.54 

12.66 

12.79 

12.91 

13.04 

4.5 

11.90 

12.03 

12.16 

12.28 

12.41 

12.53 

12.66 

12.78 

12.91 

13.03 

13.16 

4.6 

12.03 

12.15 

12.28 

12.40 

12.53 

12.65 

12.78 

12.90 

13.03 

13.15 

13.28 

4.7 

12.15 

12.27 

12.40 

12.52 

12.65 

12.77 

12.90 

13.02 

13.15 

13.27 

13.40 

4.8 

12.27 

12.39 

12.52 

12.64 

12.77 

12.89 

13.02 

13.14 

13.27 

13.39 

13.52 

4.9 

12.39 

12.51 

12.64 

12.76 

12.89 

13.01 

13.14 

13.26 

13.39 

13.51 

13.64 

5.0 

12.51 

12.63 

12.76 

12.88 

13.01 

13.13 

13.26 

13.38 

13.51 

13.63 

13.76 

5.1 

12.63 

12.75 

12.88 

13.00 

13.13 

13.25 

13.38 

13.50 

13.63 

13.76 

13.89 

5.2 

12.75 

12.87 

13.00 

13.12 

13.25 

13.37 

13.50 

13.62 

13.75 

13.88 

14.01 

5.3 

12.87 

12.99 

13.12 

13.24 

13.37 

13.49 

13.62 

13.74 

13.87 

14.00 

14.13 

5.4 

12.99 

13.11 

13.24 

13.36 

13.49 

13.61 

13.74 

13.87 

14.00 

14.12 

14.25 

5.5 

13.11 

13.23 

13.36 

13.48 

13.61 

13.73 

13.86 

13.99 

14.12 

14.24 

14.37 

5.6 

13.23 

13.35 

13.48 

13.60 

13.73 

13.86 

13.98 

14.11 

14.24 

14.36 

14.49 

5.7 

13.35 

13.47 

13.60 

13.72 

13.85 

13.98 

14.11 

14.23 

14.36 

14.48 

14.61 

5.8 

13.47 

13.59 

13.72 

13.84 

13.97 

14.10 

14.23 

14.35 

14.48 

14.61 

14.74 

5.9 

13.59 

13.71 

13.84 

13.97 

14.10 

14.22 

14.35 

14.47 

14.60 

14.73 

14.86 

APPENDIX 


211 


LXIX 

FROM  FAT  AND  LACTOMETER  READING 

AMERICAN  STANDARD 

AT  60°  F. 


31.5 

32.0 

32.5 

33.0 

33.5 

34.0 

34.5 

35.0 

35.5 

36.0 

36.5 

Fat. 

7.87 

8.00 

8.12 

8.25 

8.37 

8.50 

8.62 

8.75 

8.87 

9.00 

9.12 

0.0 

7.99 

8.12 

8.24 

8.37 

8.49 

8.62 

8.74 

8.87 

8.99 

9.12 

9.24 

0.1 

8.11 

8.24 

9.36 

8.49 

8.61 

8.74 

8.86 

8.99 

9.11 

9.24 

9.36 

0.2 

8.23 

8.36 

8.48 

8.61 

8.73 

8.86 

8.98 

9.11 

9.23 

9.36 

9.48 

0.3 

8.35 

8.48 

8.60 

8.73 

8.85 

9.98 

9.10 

9.23 

9.35 

9.48 

9.60 

0.4 

8.47 

8.60 

8.72 

8.85 

8.97 

9.10 

9.22 

9.35 

9.47 

9.60 

9.72 

0.5 

8.59 

8.72 

8.84 

8.97 

9.09 

9.22 

9.34 

9.47 

9.59 

9.72 

9.84 

0.6 

8.71 

8.84 

8.96 

9.09 

9.21 

9.34 

9.46 

9.59 

9.71 

9.84 

9.96 

0.7 

8.83 

8.96 

9.08 

9.21 

9.33 

9.46 

9.58 

9.71 

9.83 

9.96 

10.08 

0.8 

8.95 

9.08 

9.20 

9.33 

9.45 

9.58 

9.70 

9.83 

9.95 

10.08 

10.20 

0.9 

9.07 

9.20 

9.32 

9.45 

9.57 

9.70 

9.82 

9.95 

10.07 

10.20 

10.32 

1.0 

9.19 

9.32 

9.44 

9.57 

9.69 

9.82 

9.94 

10.07 

10.19 

10.32 

10.44 

1.1 

9.31 

9.44 

9.56 

9.69 

9.81 

9.94 

10.06 

10.19 

10.31 

10.44 

10.56 

.2 

9.43 

9.56 

9.68 

9.81 

9.93 

10.06 

10.18 

10.31 

10.43 

10.56 

10.68 

.3 

9.55 

9.68 

9.80 

9.93 

10.05 

10.18 

10.30 

10.43 

10.55 

10.68 

10.80 

.4 

9.67 

9.80 

9.92 

10.05 

10.17 

10.30 

10.42 

10.55 

10.67 

10.80 

10.92 

.5 

9.79 

9.92 

10.04 

10.17 

10.29 

10.42 

10.54 

10.67 

10.79 

10.92 

11.04 

.6 

9.91 

10.04 

10.16 

10.29 

10.41 

10.54 

10.66 

10.79 

10.91 

11.04 

11.16 

.7 

10.03 

10.16 

10.28 

10.41 

10.53 

10.66 

10.78 

10.91 

11.04 

11.17 

11.29 

.8 

10.15 

10.28 

10.40 

10.53 

10.65 

10.78 

10.90 

11.03 

11.16 

11.29 

11.41 

.9 

10.27 

10.40 

10.53 

10.66 

10.78 

10.91 

11.03 

11.16 

11.28 

11.41 

11.53 

2.0 

10.39 

10.52 

10.65 

10.78 

10.90 

11.03 

11.15 

11.28 

11.40 

11.53 

11.65 

2.1 

10.51 

10.64 

10.77 

10.90 

11.02 

11.15 

11.27 

11.40 

11.52 

11.65 

11.77 

2.2 

10.63 

10.76 

10.89 

11.02 

11.14 

11.27 

11.39 

11.52 

11.64 

11.77 

11.89 

2.3 

10.75 

10.88 

11.01 

11.14 

11.26 

11.39 

11.51 

11.64 

11.76 

11.89 

12.01 

2.4 

10.87 

11.00 

11.13 

11.26 

11.38 

11.51 

11.63 

11.76 

11.88 

12.01 

12.13 

2.5 

10.99 

11.12 

11.25 

11.38 

11.50 

11.63 

11.75 

11.88 

12.00 

12.13 

12.25 

2.6 

11.11 

11.24 

11.37 

11.50 

11.62 

11.75 

11.87 

12.00 

12.12 

12.25 

12.37 

2.7 

11.23 

11.37 

11.49 

11.62 

11.74 

11.87 

11.99 

12.12 

12.24 

12.37 

12.49 

2.8 

11.36 

11.49 

11.61 

11.74 

11.86 

11.99 

12.11 

12.24 

12.36 

12.47 

12.61 

2.9 

11.48 

11.61 

11.73 

11.86 

11.98 

12.11 

12.23 

12.36 

12.49 

12.61 

12.74 

3.0 

11   60 

11.73 

11.85 

11.98 

12.10 

12.23 

12.35 

12.48 

12.61 

12.74 

12  86 

3   1 

11.72 

11.85 

11.97 

12.10 

12.22 

12.35 

12.48 

12.61 

12.73 

12.86 

12.98 

3.2 

11.84 

11.97 

12.09 

12.22 

12.35 

12.48 

12.60 

12.73 

12.85 

12.98 

13.10 

3.3 

11.96 

12.09 

12.21 

12.34 

12.47 

12.60 

12.72 

12.85 

12.97 

13.10 

13.22 

3.4 

12.08 

12.21 

12.33 

12.46 

12.59 

12.72 

12.84 

12.97 

13.09 

13.22 

13.34 

3.5 

12.20 

12.33 

12.45 

12.58 

12.71 

12.84 

12.96 

13.09 

13.21 

13.34 

13.46 

3.6 

12.32 

12.45 

12.57 

12.70 

12.83 

12.96 

13.08 

13.21 

13.33 

13.46 

13.58 

3.7 

12.44 

12.57 

12.69 

12.82 

12.95 

13.08 

13.20 

13.33 

13.45 

13.58 

13.70 

3.8 

12.56 

12.69 

12.81 

12.94 

13.07 

13.20 

13.32 

13.45 

13.57 

13.70 

13.83 

3.9 

12.68 

12.81 

12.93 

13.06 

13.19 

13.32 

13.44 

13.57 

13.70 

13.83 

13.95 

4.0 

12.80 

12.93 

13.05 

13.18 

13.31 

13.44 

13.56 

13.69 

13.82 

13.95 

14.07 

4.1 

12.92 

13.05 

13.18 

13.31 

13.43 

13.56 

13.69 

13.82 

13.94 

14.07 

14.19 

4.2 

13.05 

13.18 

13.30 

13.43 

13.55 

13.68 

13.81 

13.94 

14.06 

14.19 

14.31 

4.3 

13.17 

13.30 

13.42 

13.55 

13.67 

13.80 

13.93 

14.06 

14.18 

14.31 

14.43 

4.4 

13.29 

13.42 

13.54 

13.67 

13.79 

13.92 

14.05 

14.18 

14.30 

14.43 

14.55 

4.5 

13.41 

13.54 

13.66 

13.79 

13.91 

14.04 

14.17 

14.30 

14.42 

14.55 

14.67 

4.6 

13.53 

13.66 

13.78 

13.91 

14.03 

14.16 

14.29 

14.42 

14.54 

14.67 

14  79 

4.7 

13.65 

13.78 

13.90 

14.03 

14.15 

14.28 

14.41 

14.54 

14.66 

14.79 

14.91 

4.8 

13.77 

13.90 

14.02 

14.15 

14.27 

14.40 

14.53 

14.66 

14.78 

14.91 

15.03 

4.9 

13.89 

14.02 

14.14 

14.27 

14.39 

14.52 

14.65 

14.78 

14.90 

15.03 

15.15 

5.0 

14.01 

14.14 

14.26 

14.39 

14.51 

14.64 

14.77 

14.90 

15.02 

15.15 

15.27 

5.1 

14.13 

14.26 

14.38 

14.51 

14.63 

14.76 

14.89 

15.02 

15.14 

15.27 

15.39 

5.2 

14.25 

14.38 

14.50 

14.63 

14.75 

14.88 

15.01 

15.14 

15.26 

15.39 

15.51 

5.3 

14.37 

14.50 

14.62 

14.75 

14.88 

15.01 

15.13 

15.26 

15.38 

15.51 

15.63 

5.4 

14.49 

14.62 

14.75 

14.87 

15.00 

15.13 

15.25 

15.38 

15.50 

15.63 

15.75 

5.5 

14.61 

14.75 

14.87 

14.99 

15.12 

15.25 

15.37 

15.50 

15.62 

15.75 

15  87 

5.6 

14.74 

14.87 

14.99 

15.11 

15.24 

15.37 

15.49 

15.  -62 

15.74 

15.87 

15.99 

5.7 

14.86 

14.99 

15.11 

15.23 

15.36 

15.49 

15.61 

15.74 

15.86 

15.99 

16.12 

5.8 

14.98 

15.11 

15.23 

15.36 

15.48 

15.61 

15.73 

15.86 

15.99 

16.12 

16.24 

5.9 

212 


APPENDIX 


TABLE 

FOR  CALCULATING  TOTAL  SOLIDS  FROM 

ACCORDING  TO 


a 

LACTOMETER  READING 

£ 

26.0 

26.5 

27.0 

27.5 

28.0 

28.5 

29.0 

29.5 

30.0 

30.5 

31.0 

0.0 

6.652 

6.776 

6.900 

7  .  025 

7.150 

7.274 

7.397 

7.522 

7.647 

7.771 

7.895 

0.1 

6.77 

6.90 

7.02 

7.15 

7.27 

7.39 

7.52 

7.64 

7.77 

7.89 

8.02 

0.2 

6.89 

7.02 

7.14 

7.26 

7.39 

7.51 

7.64 

7.76 

7.89 

8.01 

8.14 

0.3 

7.01 

7.14 

7.26 

7.39 

7.51 

7.63 

7.76 

7.88 

8.01 

8.13 

8.26 

0.4 

7.13 

7.26 

7.38 

7.51 

7.63 

7.75 

7.88 

8.00 

8.13 

8.25 

8.38 

0.5 

7.25 

7.38 

7.50 

7.63 

7.75 

7.87 

8.00 

8.12 

8.25 

8.37 

8.50 

0.6 

7.37 

7.50 

7.62 

7.75 

7.87 

7.99 

8.12 

8.24 

8.37 

8.49 

8.62 

0.7 

7.49 

7.62 

7.74 

7.87 

7.99 

8.11 

8.24 

8.36 

8.49 

8.61 

8.74 

0.8 

7.61 

7.74 

7.86 

7.99 

8.11 

8.23 

8.36 

8.48 

8.61 

8.73 

8.86 

0.9 

7.73 

7.86 

7.98 

8.11 

8.23 

8.35 

8.48 

8.60 

8.73 

8.85 

8.98 

.0 

7.85 

7.98 

8.10 

8.23 

8.35 

8.47 

8.60 

8.72 

8.85 

8.97 

9.10 

.1 

7.97 

8.10 

8.22 

8.35 

8.47 

8.59 

8.72 

8.84 

8.97 

9.09 

9.22 

.2 

8.09 

8.22 

8.34 

8.47 

8.59 

8.71 

8.84 

8.96 

9.09 

9.21 

9.34 

.3 

8.21 

8.34 

8.46 

8.59 

8.71 

8.83 

8.96 

9.08 

9.21 

9.33 

9.46 

.4 

8.33 

8.46 

8.58 

8.71 

8.83 

8.95 

9.08 

9.20 

9.33 

9.45 

9.58 

.5 

8.45 

8.58 

8.70 

8.83 

8.95 

9.07 

9.20 

9.32 

9.45 

9.57 

9.70 

.6 

8.57 

8.70 

8.82 

8.95 

9.07 

9.19 

9.32 

9.44 

9.57 

9.69 

9.82 

.7 

8.69 

8.82 

8.94 

9.07 

9.19 

9.31 

9.44 

9.56 

9.69 

9.81 

9.94 

.8 

8.81 

8.94 

9.06 

9.19 

9.31 

9.43 

9.56 

9.68 

9.81 

9.93 

10.06 

.9 

8.93 

9.06 

9.18 

9.31 

9.43 

9.55 

9.68 

9.80 

9.93 

10.05 

10.18 

2.0 

9.05 

9.18 

9.30 

9.43 

9.55 

9.67 

9.80 

9.92 

10.05 

10.17 

10.30 

2.1 

9.17 

9.30 

9.42 

9.55 

9.67 

9.79 

9.92 

10.04 

10.17 

10.29 

10.42 

2.2 

9.29 

9.42 

9.54 

9.67 

9.79 

9.91 

10.04 

10.16 

10.29 

10.41 

10.54 

2.3 

9.41 

9.54 

9.66 

9.79 

9.91 

10.03 

10.16 

10.28 

10.41 

10.53 

10.66 

2.4 

9.53 

9.66 

9.78 

9.91 

10.03 

10.15 

10.28 

10.40 

10.53 

10.65 

10.78 

2.5 

9.65 

9.78 

9.90 

10.03 

10.15 

10.27 

10.40 

10.52 

10.65 

10.77 

10.90 

2.6 

9.77 

9.90 

10.02 

10.15 

10.27 

10.39 

10.52 

10.64 

10.77 

10.89 

11.02 

2.7 

9.89 

10.02 

10.14 

10.27 

10.39 

10.51 

10.64 

10.76 

10.89 

11.01 

11.14 

2.8 

10.01 

10.14 

10.26 

10.39 

10.51 

10.63 

10.76 

10.88 

11.01 

11.13 

11.26 

2.9 

10.13 

10.26 

10.38 

10.51 

10.63 

10.75 

10.88 

11.00 

11.13 

11:25 

11.38 

3.0 

10.25 

10.38 

10.50 

10.63 

10.75 

10.87 

11.00 

11.12 

11.25 

11.37 

11.50 

3.1 

10.37 

10.50 

10.62 

10.75 

10.87 

10.99 

11.12 

11.24 

11.37 

11.49 

11.62 

3.2 

10.49 

10.62 

10.74 

10.87 

10.99 

11.11 

11.24 

11.36 

11.49 

11.61 

11.74 

3.3 

10.61 

10.74 

10.86 

10.99 

11.11 

11.23 

11.36 

11.48 

11.61 

11.73 

11.86 

3.4 

10.73 

10.86 

10.98 

11.11 

11.23 

11.35 

11.48 

11.60 

11.73 

11.85 

11.98 

3.5 

10.85 

10.98 

11.10 

11.23 

11.35 

11.47 

11.60 

11.72 

11.85 

11.97 

12.10 

3.6 

10.97 

11.10 

11.22 

11.35 

11.47 

11.59 

11.72 

11.84 

11.97 

12.09 

12.22 

3.7 

11.09 

11.22 

11.34 

11.47 

11.59 

11.71 

11.84 

11.96 

12.09 

12.21 

12.34 

3.8 

11.21 

11.34 

11.46 

11.59 

11.71 

11.83 

11.96 

12.08 

12.21 

12.33 

12.46 

3  9 

11  33 

11  46 

11.58 

11  71 

11  83 

11  95 

12  08 

12.20 

12  33 

12  45 

1?  58 

4.0 

11.45 

11.58 

11.70 

11.83 

11.95 

12.07 

12.20 

12.32 

12.45 

12.57 

12.70 

4   1 

11   57 

11.70 

11.82 

11.95 

12.07 

12.19 

12.32 

12.44 

12.57 

12.69 

1?  82 

4.2 

11.69 

11.82 

11.94 

12.07 

12.19 

12.31 

12.44 

12.56 

12.69 

12.81 

12.94 

4.3 

11.81 

11.94 

12.06 

12.19 

12.31 

12.43 

12.56 

12.68 

12.81 

12.93 

13.06 

4.4 

11.93 

12.06 

12.18 

12.31 

12.43 

12.55 

12.68 

12.80 

12.93 

13.05 

13.18 

4.5 

12.05 

12.18 

12.30 

12.43 

12.55 

12.67 

12.80 

12.92 

13.05 

13.17 

13.30 

4.6 

12.17 

12.30 

12.42 

12.55 

12.67 

12.79 

12.92 

13.04 

13.17 

13.29 

13.42 

4.7 

12.29 

12.42 

12.54 

12.67 

12.79 

12.91 

13.04 

13.16 

13.29 

13.41 

13.54 

4.8 

12.41 

12.54 

12.66 

12.79 

12.91 

13.03 

13.16 

13.28 

13.41 

13.53 

13.66 

4.9 

12.53 

12.66 

12.78 

12.91 

13.03 

13.15 

13.28 

13.40 

13.53 

13.65 

13.78 

5.0 

12.65 

12.78 

12.90 

13.03 

13.15 

13.27 

13.40 

13.52 

13.65 

13.77 

13.90 

5.1 

12.77 

12.90 

13.02 

13.15 

13.27 

13.39 

13.52 

13.64 

13.77 

13.89 

14.02 

5.2 

12.89 

13.02 

13.14 

13.27 

13.39 

13.51 

13.64 

13.76 

13.89 

14.01 

14.14 

5.3 

13.01 

13.14 

13.26 

13.39 

13.51 

13.63 

13.76 

13.88 

14.01  i 

14.13 

14.26 

5.4 

13.13 

13.26 

13.38 

13.51 

13.63 

13.75 

13.88 

14.00 

14.13 

14.25 

14.38 

5.5 

13.25 

13.38 

13.50 

13.63 

13.75 

13.87 

14.00 

14.12 

14.25 

14.37 

14.50 

5.6 

13.37 

13.50 

13.62 

13.75 

13.87 

13.99 

14.12 

14.24 

14.37 

14.49 

14.62 

5.7 

13.49 

13.62 

13.74 

13.87 

13.99 

14.11 

14.24 

14.36 

14.49  / 

14.61 

14.74 

5.8 

13.61 

13.74 

13.86 

13.99 

14.11 

14.23 

14.36 

14.48 

14.61 

14.73 

14.86 

5.9 

13.73 

13.86 

13.98 

14.11 

14.23 

14.35 

14.48 

14.60 

14.73 

14.85 

14.98' 

APPENDIX 


213 


LXX 

FAT  AND  LACTOMETER  READING 
DROOP  RICHMOND 


AT    60° 

F. 

v 

« 

31.5 

32.0 

32.5 

33.0 

33.5 

34.0 

34.5 

35.0 

35.5 

36.0 

36.5 

£ 

8.018 

8.140 

8.264 

8.387 

8.509 

-8.631 

8.755 

8.878 

9.000 

9.122 

9.244 

0.0 

8.14 

8.26 

8.38 

8.51 

8.63 

8.75 

8.88 

9.00 

9.12 

9.24 

9.36 

0.1 

8.26 

8.38 

8.50 

8.63 

8.75 

8.87 

9.00 

9.12 

9.24 

9.36 

9.48 

0.2 

8.38 

8.50 

8.62 

8.75 

8.87 

8.99 

9.12 

9.24 

9.36 

9.48 

9.60 

0.3 

8.50 

8.62 

8.74 

8.87 

8.99 

9.11 

9.24 

9.36 

9.48 

9.60 

9.72 

0  4 

8.62 

8.74 

8.86 

8.99 

9.11 

9.23 

9.36 

9.48 

9.60 

9.72 

9.84 

0.5 

8.74 

8.86 

8.98 

9.11 

9.23 

9.35 

9.48 

9.60 

9.72 

9.84 

9.96 

0  6 

8.86 

8.98 

9.10 

9.23 

9.35 

9.47 

9.60 

9.72 

9.84 

9.96 

10.08 

0.7 

8.98 

9.10 

9.22 

9.35 

9.47 

9.59 

9.72 

9.84 

9.96 

10.08 

10.20 

0.8 

9.10 

9.22 

9.34 

9.47 

9.59 

9.71 

9.84 

9.96 

10.08 

10.20 

10.32 

0.9 

9.22 

9.34 

9.46 

9.59 

9.71 

9.83 

9.96 

10.08 

10.20 

10.32 

10.44 

.0 

9.34 

9.46 

9.58 

9.71 

9.83 

9.95 

10.08 

10.20 

10.32 

10.44 

10.56 

.1 

9.46 

9.58 

9.70 

9.83 

9.95 

10.07 

10.20 

10.32 

10.44 

10.56 

10.68 

.2 

9.58 

9.70 

9.82 

9.95 

10.07 

10.19 

10.32 

10.44 

10.56 

10.68 

10.80 

.3 

9.70 

9.82 

9.94 

10.07 

10.19 

10.31 

10.44 

10.56 

10.68 

10.80 

10.92 

.4 

9.82 

9.94 

10.06 

10.19 

10.31 

10.43 

10.56 

10.68 

10.80 

10.92 

11.04 

5 

9.94 

10.06 

10.18 

10.31 

10.43 

10.55 

10.68 

10.80 

10.92 

11.04 

11.16 

.6 

10.06 

10.18 

10.30 

10.43 

10.55 

10.67 

10.80 

10.92 

11.04 

11.16 

11.28 

.7 

10.18 

10.30 

10.42 

10.55 

10.67 

10.79 

10.92 

11.04 

11.16 

11.28 

11.40 

.8 

10.30 

10.42 

10.54 

10.67 

10.79 

10.91 

11.04 

11.16 

11.28 

11.40 

11.52 

.9 

10.42 

10.54 

10.66 

10.79 

10.91 

11.03 

11.16 

11.28 

11.40 

11.52 

11.64 

2.0 

10.54 

10.66 

10.78 

10.91 

11.03 

11.15 

11.28 

11.40 

11.52 

11.64 

11.76 

2.1 

10.66 

10.78 

10.90 

11.03 

11.15 

11.27 

11.40 

11.52 

11.64 

11.76 

11.88 

2.2 

10.78 

10.90 

11.02 

11.15 

11.27 

11.39 

11.52 

11.64 

11.76 

11.88 

12.00 

2.3 

10.90 

11.02 

11.14 

11.27 

11.39 

11.51 

11.64 

11.76 

11.88 

12.00 

12   12 

2.4 

11.02 

11.14 

11.26 

11.39 

11.51 

11.63 

11.76 

11.88 

12.00 

12.12 

Az.24 

2.5 

11.14 

11.26 

11.38 

11.51 

11.63 

11.75 

11.88 

12.00 

12.12 

12.24 

12.36 

2.6 

11.26 

11.38 

11.50 

11.63 

11.75 

11.87 

12.00 

12.12 

12.24 

12.36 

12.48 

2.7 

11.38 

11.50 

11.62 

11.75 

11.87 

11.99 

12.12 

12.24 

12.36 

12.48 

12.60 

2.8 

11.50 

11.62 

11.74 

11.87 

11.99 

12.11 

12.24 

12.36 

12.48 

12.60 

12.72 

2.9 

11.62 

11.74 

11.86 

11.99 

12.11 

12.23 

12.36 

12.48 

12.60 

12.72 

12.84 

3.0 

11.74 

11.86 

11.98 

12.11 

12.22 

12.35 

12.48 

12.60 

12.72 

12.84 

12.96 

3.1 

11.86 

11.98 

12.10 

12.23 

12.35 

12.47 

12.60 

12.72 

12.84 

12.96 

13.08 

3.2 

11.98 

12.10 

12.22 

12.35 

12.47 

12.59 

12.72 

12.84 

12.96 

13.08 

13.20 

3.3 

12.10 

12.22 

12.34 

12.47 

12.59 

12.71 

12.84 

12.96 

13.08 

13.20 

13.32 

3.4 

12.22 

12.34 

12.46 

12.59 

12.71 

12.83 

12.96 

13.08 

13.20 

13.32 

13.44 

3.5 

12.34 

12.46 

12.58 

12.71 

12.83 

12.95 

13.08 

13.20 

13.32 

13.44 

13.56 

3.6 

12.46 

12.58 

12.70 

12.83 

12.95 

13.07 

13.20 

13.32 

13.44 

13.56 

13.68 

3.7 

12.58 

12.70 

12.82 

12.95 

13.07 

13.19 

13.32 

13.44 

13.56 

13.68 

13.80 

3.8 

12.70 

12.82 

12.94 

13.07 

13.19 

13.31 

13.44 

13.56 

13.68 

13.80 

13.92 

3.9 

12.82 

12.94 

13.06 

13.19 

13.31 

13.43 

13.56 

13.68 

13.80 

13.92 

14.04 

4.0 

12.94 

13.06 

13.18 

13.31 

13.43 

13.55 

13.68 

13.80 

13.92 

14.04 

14.16 

4.1 

13.06 

13.18 

13.30 

13.43 

13.55 

13.67 

13.80 

13.92 

14.04 

14.16 

14.28 

4.2 

13.18 

13.30 

13.42 

13.55 

13.67 

13.79 

13.92 

14.04 

14.16 

14.28 

14.40 

4.3 

13.30 

13.42 

13.54 

13.67 

13.79 

13.91 

14.04 

14.16 

14.28 

14.40 

14.52 

4.4 

13.42 

13.54 

13.66 

13.79 

13.91 

14.02 

14.16 

14.28 

14.40 

14.52 

14.64 

4.5 

13.54 

13.66 

13.78 

13.91 

14.03 

14.15 

14.28 

14.40 

14.52 

14.64 

14.76 

4.6 

13.66 

13.78 

13.90 

14.03 

14.15 

14.27 

14.40 

14.52 

14.64 

14.76 

14.88 

4.7 

13.78 

13.90 

14.02 

14.15 

14.27 

14.39 

14.52 

14.64 

14.76. 

14.88 

15.00 

4.8 

13.90 

14.02 

14.14 

14.27 

14.39 

14.51 

14.64 

14.76 

14.88 

15.00 

15.12 

4.9 

14.02 

14.14 

14.26 

14.39 

14.51 

14.63 

14.76 

14.88 

15.00 

15.12 

15.24 

5.0 

14.14 

14.26 

14.38 

14.51 

14.63 

14.75 

14.88 

15.00 

15.12 

15  .  24 

15.36 

5.1 

14.26 

14.38 

14.50 

14.63 

14.75 

14.87 

15.00 

15.12 

15.24 

15.36 

15.48 

5.2 

14.38 

14.50 

14.62 

14.75 

14.87 

14.99 

15.12 

15.24 

15.36 

15.48 

15.60 

5.3 

14.50 

14.62 

14.74 

14.87 

14.99 

15.11 

15.24 

15.36 

15.48 

15.60 

15.72 

5.4 

14.62 

14.74 

14.86 

14.99 

15.11 

15.23 

15.36 

15.48 

15.60 

15.72 

15.84 

5.5 

14.74 

14.86 

14.98 

15.11 

15.23 

15.35 

15.48 

15.60 

15.72 

15.84 

15.96 

5.6 

14.86 

14.98 

15.10 

15.23 

15.35 

15.47 

5.60 

15.72 

15.84 

15.96 

16.08 

5.7 

14.98 

15.10 

15.22 

15.35 

15.47 

15.59 

5.72 

15.84 

15.96 

16.08 

16.20 

5.8 

15.10 

15.22 

15.34' 

15.47 

15.59 

15.71 

5.84 

15.96 

16.08 

16.20 

16.32 

5.9 

214 


APPENDIX 


TABLE  LXXI 

TABLE  FOR  CONVERSION  OF  CUPROUS  OXIDE  (Cu2O)  AND 

COPPER   TO  LACTOSE 

MILLIGRAMS 


CuzO 

Cu 

Lactose 

CvuO 

Cu 

Lactose 

Cu20 

Cu 

Lactose 

112.6 

100 

71.6 

157.6 

140 

101.3 

202.7 

180 

131.6 

113.7 

101 

72.4 

158.7 

141 

102.0 

203.8 

181 

132.4 

114.8 

102 

73.1 

159.8 

142 

102.8 

204.9 

182 

133.1 

115.9 

103 

73.8 

160.9 

143 

103.5 

206.0 

183 

133.9 

117.0 

104 

74.6 

162.0 

144 

104.3 

207.1 

184 

134.7 

118.2 

105 

75.3 

163.2 

145 

105.1 

208.3 

185 

135.4 

119.3 

106 

76.1 

164.3 

146 

105.8 

209.4 

186 

136.2 

120.4 

107 

76.8 

165.5 

147 

106.6 

210.5 

187 

137.0 

121.5 

108 

77.6 

166.6 

148 

107.3 

211.6 

188 

137.7 

122.7 

109 

78.3 

167.7 

149 

108.1 

212.7 

189 

138.5 

123.8 

110 

79.0 

168.9 

150 

108.8 

213.9 

190 

139.3 

124.9 

111 

79.8 

170.0 

151 

109.6 

215.0 

191 

140.0 

126.0 

112 

80.5 

171.1 

152 

110.3 

216.1 

192 

140.8 

127.1 

113 

81.3 

172.2 

153 

111.1 

217.2 

193 

141.6 

128.2 

114 

82.0 

173.3 

154 

111.9 

218.3 

194 

142.3 

129.4 

115 

82.7 

174.5 

155 

112.6 

219.5 

195 

143.1 

130.5 

116 

83.5 

175.6 

156 

113.4 

220.6 

196 

143.9 

131.7 

117 

84.2 

176.7 

157 

114.1 

221.8 

197 

144.6 

132.8 

118 

85.0 

177.8 

158 

114.9 

222.9 

198 

145.4 

133.9 

119 

85.7 

178.9 

159 

115.6 

224.0 

199 

146.2 

135.1 

120 

86.4 

180.1 

160 

116.4 

225.2 

200 

146.9 

136.2 

121 

87.2 

181.2 

161 

117.1 

226.3 

201 

147.7 

137.3 

122 

87.9 

182.3 

162 

117.9 

227.4 

202 

148.5 

138.4 

123 

88.7 

183.4 

163 

118.6 

228.5 

203 

149.2 

139.5 

124 

89.4 

184.5 

164 

119.4 

229.6 

204 

150.0 

140.7 

125 

90.1 

185.7 

165 

120.2 

230.7 

205 

150.7 

141.8 

126 

90.9 

186.8 

166 

120.9 

231.9 

206 

151.5 

143.0 

127 

91.6 

188.0 

167 

121.7 

233.0 

207 

152.2 

144.1 

128 

92.4 

189.1 

168 

122.4 

234.1 

208 

153.0 

145.2 

129 

93.1 

190.2 

169 

123.2 

235.2 

209 

153.7 

146.4 

130 

93.8 

191.4 

170 

123.9 

236.4 

210 

154.5 

147.5 

131 

94.6 

192.5 

171 

124.7 

237.5 

211 

155.2 

148.6 

132 

95.3 

193.6 

172 

125.5 

238.6 

212 

156.0 

149.7 

133 

96.1 

194.7 

173 

126.2 

239.7 

213 

156.7 

150.8 

134 

96.9 

195.8 

174 

127.0 

240.8 

214 

157.5 

152.9 

135 

97.6 

197.0 

175 

127.8 

242.0 

215 

158.2 

153.1 

136 

98.3 

198.1 

176 

128.5 

243.1 

216 

159.0 

154.2 

137 

99.1 

199.3 

177 

129.3 

244.3 

217 

159.7 

155.3 

138 

99.8 

200.4 

178 

130.1 

245.4 

218 

160.4 

156.4 

139 

100.5 

201.5 

179 

130.8 

246.5 

219 

161.2 

APPENDIX 


215 


TABLE  LXXI— Continued 

TABLE  FOR  CONVERSION  OF  CUPROUS   OXIDE   (Cu2O)  AND 

COPPER  TO  LACTOSE 

MILLIGRAMS 


Cu2O 

Cu 

Lactose 

Cu20 

Cu 

Lactose 

Cu2O 

Cu 

Lactose 

247.7 

220 

161.9 

292.7 

260 

192.5 

337.8 

300 

224.4 

248.8 

221 

162.7 

293.8 

261 

193.3 

338.9 

301 

225.2 

249.9 

222 

163.4 

294.9 

262 

194.1 

340.0 

302 

225.9 

251.0 

223 

164.2 

296.0 

263 

194.9 

341.1 

303 

226.7 

252.1 

224 

164.9 

297.1 

264 

195.7 

342.2 

304 

227.5 

253.3 

225 

165.7 

298.3 

265 

196.4 

343.4 

305 

228.3 

254.4 

226 

166.4 

299.4 

266 

197.2 

344.5 

306 

229.1 

255.5 

227 

167.2 

300.5 

267 

198.0 

345.6 

307 

229.8 

256.6 

228 

167.9 

301.6 

268 

198.8 

346.7 

308 

230.6 

257.7 

229 

168.6 

302.7 

269 

199.5 

347.8 

309 

231.4 

258.9 

230 

169.4 

303.9 

270 

200.3 

349.0 

310 

232.2 

260.0 

231 

170.1 

305.0 

271 

201.1 

350.1 

311 

232.9 

261.1 

232 

170.9 

306.2 

272 

201.9 

351.2 

312 

233.7 

262.2 

233 

171.6 

307.3 

273 

202.7 

352.3 

313 

234.5 

263.3 

234 

172.4 

308.4 

274 

203.5 

353.4 

314 

235.3 

264.5 

235 

173.1 

309.6 

275 

204.3 

354.6 

315 

236.1 

265.6 

236 

173.9 

310.7 

276 

205.1 

355.7 

316 

236.8 

266.8 

237 

174.6 

311.8 

277 

205.9 

356.8 

317 

237.6 

267.9 

238 

175.4 

313.0 

278 

206.7 

357.9 

318 

238.4 

269.0 

239 

176.2 

314.1 

279 

207.5 

359.0 

319 

239.2 

270.2 

240 

176.9 

315.3 

280 

208.3 

360.2 

320 

240.0 

271.3 

241 

177.7 

316.4 

281 

209.1 

361.3 

321 

240.7 

272.4 

242 

178.5 

317.5 

282 

209.9 

362.4 

322 

241.5 

273.5 

243 

179.3 

318.6 

283 

210.7 

363.5 

323 

242.3 

274.6 

244 

180.1 

319.7 

284 

211.5 

364.6 

324 

243.1 

275.8 

245 

180.8 

320.9 

285 

212.3 

365.8 

325 

243.9 

276.9 

246 

181.6 

322.0 

286 

213.1 

366.9 

326 

244.6 

278.1 

247 

182.4 

323.1 

287 

213.9 

368.0 

327 

245  .4 

279.2 

248 

183.2 

324.2 

288 

214.7 

369.1 

328 

246.2 

280.3 

249 

184.0 

325.3 

289 

215.5 

370.2 

329 

247.0 

281.5 

250 

184.4 

326.5 

290 

216.3 

371.4 

330 

247.7 

282.6 

251 

185.5 

327.6 

291 

217.1 

372.5 

331 

248.5 

283.7 

252 

186.3 

328.7 

292 

217.9 

373.6 

332 

249.2 

284.8 

253 

187.1 

329.8 

293 

218.7 

374.7 

333 

250.0 

286.0 

254 

187.9 

330.9 

294 

219.5 

375.8 

334 

250.8 

287.1 

255 

188.7 

332.1 

295 

220.3 

377.0 

335 

251.6 

288.2 

256 

189.4 

333.2 

296 

221.1 

378.1 

336 

252.5 

289.3 

257 

190.2 

334.4 

297 

221.9 

379.3 

337 

253.3 

290.4 

258 

191.0 

335.5 

298 

222.7 

380.4 

338 

254.1 

291.5 

259 

191.8 

336.7 

299 

223.5 

381.5 

339 

254.9 

216 


APPENDIX 


TABLE  LXXI — Continued 

TABLE  FOR  CONVERSION  OF  CUPROUS  OXIDE   (Cu2O)  AND 
COPPER  TO  LACTOSE 

MILLIGRAMS 


CuaO 

Cu 

Lactose 

Cu20 

Cu 

Lactose 

Cu2O 

Cu 

Lactose 

382.7 

340 

255.7 

405.3 

360 

272.1 

427.9 

380 

289.1 

383.8 

341 

256.5 

406.4 

361 

272.9 

429.0 

381 

289.9 

385.0 

342 

257.4 

407.5 

362 

273.7 

430.1 

382 

290.8 

386.1 

343 

258.2 

408.6 

363 

274.5 

431.2 

383 

291.7 

387.2 

344 

259.0 

409.7 

364 

275.3 

432.3 

384 

292.5 

388.4 

345 

259.8 

410.9 

365 

276.2 

433.5 

385 

293.4 

389.5 

346 

260.6 

412.0 

366 

277.1 

434.6 

386 

294.2 

390.6 

347 

261.4 

413.1 

367 

277.9 

435.8 

387 

295.1 

391.7 

348 

262.3 

414.2 

368 

278.8 

436.9 

388 

296.0 

392.8 

349 

263.1 

415.3 

369 

279.6 

438.0 

389 

296.8 

394.0 

350 

263.9 

416.5 

370 

280.5 

439.2 

390 

297.7 

395.1 

351 

264.7 

417.6 

371 

281.4 

440.3 

391 

298.5 

396.2 

352 

265.5 

418.8 

372 

282.2 

441.4 

392 

299.4 

397.3 

353 

266.3 

419.9 

373 

283.1 

442.5 

393 

300.3 

398.4 

354 

267.2 

421.0 

374 

283.9 

443.6 

394 

301.1 

399.6 

355 

268.0 

422.2 

375 

284.8 

444.8 

395 

302.0 

400.7 

356 

268.8 

423.3 

376 

285.7 

445.9 

396 

302.8 

401.9 

357 

269.6 

424.5 

377 

286.5 

447.0 

397 

303.7 

403.0 

358 

270  .4 

425.6 

378 

287.4 

448.1 

398 

304.6 

404.1 

359 

271.2 

426.7 

379 

288.2 

449.2 

399 

305.4 

450.4 

400 

306.3 

SUBJECT  INDEX 


Abnormal  milk,  54 
Acidity,  75 

and  bacteria,  132 

of  media,  119,  121 
Acid  producing  organisms,  191 
Aciduric  bacilli,  195 
Adulteration  of  milk,  55 

calculation  of,  58 
Agar  media,  117,  120 

whey,  119 

lactose,  119 

lactose  bile  salt,  143 

sesculin,  143 

casein,  194,  208 
iggressins,  27 
Air,  bacteria  in,  100 
Albumin,  74 

effect  of  heat  on,  189 
Aldehyde  value,  75 
Alkali-forming  organisms,  194 
Ambocepters,  26 
Amylase,  22 

detection  and  estimation,  91 
Aniline  orange,  86 
Annatto,  86 
Antibodies,  26 

"Appeal  to  the  cow"  test,  59 
Ash,  50,  76 

estimation  of,  69 

B 

B.  abortus,  190 

characteristics  of,  192 
B.  bulgaricus,  196 


B.  butyricus,  147 

B.  coli,  136 

appearance  of  colonies,  144 
calculation  of  results,  142 
effect  of    atmospheric    tempera- 
ture, 139 

enrichment  methods,  140 
estimation  of,  140 
grain  types,  145 
liquid  media  for,  140 
plate  methods  of  estimating,  143 
rate  of  development,  107 
type,  classification  of,  145 

B.  diphtheria,  156 
detection  of,  157 

B.  enteritidis  sporogenes,  146 

B.  lactis  acidi,  109 

B.  lactis  aerogenes,  109,  119 

B.  paratyphosus,  161 

B.  tuberculosis,  135 
detection  of,  164 
inoculation  method,  165 
pseudo,  168 
types,  169 

B.  typhosus,  159 
.  isolation  of,  160 

Bacteria  in  milk,  93 
acid-producing,  106 
alkali-producing,  106 
development  of,  102 
effect  of  brushing  cows  on,  98 
effect  of  low  temperatures  on,  111 
enumeration  of,  113 
Breed's  method,  129 
by  acidity,  132 
217 


218 


SUBJECT  INDEX 


Bacteria  in  milk,  enumeration,  of, 

direct  methods,  126 
methylene  blue  test,  130 
plate  methods,  116 
intra-mammary,  93 
Bacterial  counts,  accuracy  of.  117, 

121 

effect  of  sugars  on,  118 
Benzoic  acid,  84 
Borates,  83 
Boric  acid,  83 
Breed  of  cattle,  37 

effect  on  fat  constants,  38 
effect  on  milk  composition,  47 

C 

Cane  sugar,  88 
Caramel,  86 
Caseinogen,  7 

composition  of,  8 

estimation  of,  74 

hydrolysis  of,  11 

meta,  7 

para,  14 

properties  of,  10 

reaction  with  rennin,  13,  16 
Catalase,  23 

estimation  of,  91 
Cells,  171 

blood,  173 

epithelial,  172 

estimation  of,  174 

foam,  173 

number  in  milk,  178 
Certified  milk,  138 
Colonies,  counting  of,  125 
Colostrum,  52 
Colouring  matter,  85 
Complement,  26 
Composition  of  milk,  34 

limits  of,  37 

maximum  variations,  35 

variations,  37 


Condensed  milk,  88 

Conductivity,  31 

Containers,  Bacteria  in  milk,  100 

Coolers,  100 

Counting  lens,  126 

Cream,  87 

line  hi  pasteurised  milk,  185 
Curd  test,  197 

bacterial  flora,  200 

types,  198 

D 

Death  points  in  milk: 

B.  diphtheria,  187 

B.  tuberculosis,  187 

B.  typhosus,  187 
Debris,  161 

estimation  of,  180 
Diphtheroid  bacilli,  158 
Dirt,  161 

estimation  of,  180 

significance  of,  183 

testers,  182 
Disease,  effect  on  composition,  54 

E 

Enrichment  methods  for  B.  coli,  140 
Enzymes,  21 

effect  of  heat  on,  186 

estimation  of,  88 
Epithelial  cells,  172 
Erythrocytes,  173 
Excremental  organisms,  135 


Fat,  constants  of,  2 

estimation  of,  66 

globules,  1,  44,  52 

nature  of,  1 
Fermentation  test,  197 
Food,    effect    on    composition    of 
milk,  39 

bacteria  in,  99 


SUBJECT  INDEX 


219 


Fore  milk,  50 

bacteria  in,  96 
Formaldehyde,  81 
Freezing  point  of  milk,  30 


Galactase,  22 

estimation  of,  92 
Gaertner  group,  161 
Gases  in  milk,  21 
Gelatine,  detection  of,  87 

media,  117,  120 
Germicidal  action,  102 

H 

Haemolysins,  27 

Haemolytic  streptococci,  151 

Hoffman's  bacillus,  158 

Homogenised  milk,  30 

Hypochlorites,  85 

Hydrogen  ion  concentration,  121 

Hydrogen  peroxide,  85 


Immune  bodies,  24 
Incubation  period,  117 
Inert  organisms,  194 
Intra-mammary  bacterial  pollution, 
93 


Lactalbumin,  17,  74 

properties  of,  18 
Lactation  stage,  effect  of,  45,  49 
Lacto  globulin,  18 
Lactokinase,  22 
Lactometer  table,  209 
Lactose,  bile,  140 

broth,  140 

estimation  of,  71 

origin  of,  3 

properties,  5 

specific  rotation,  9 

table,  214 


Lecithin,  20 
Leucocytes,  173 
Lipase,  22 
Litter,  bacteria  in,  99 

M 

Media,  acidity  of,  119 

SBSculin,  143,  207 

brilliant  green,  160 

casein,  208 

Drigalski  and  Conradi's,  143 

egg,  169,  208 

Endo's,  143 

for  B.  coli,  141 

rebipelagar,  143,  207 

standard,  120,  122 
Methyl  red  reaction,  145 
Milk  coolers,  effect  of,  100 
Milking  intervals,  effect  of,  42 
Milk  serum,  78 
Mineral  constituents,  76 
Morgan's  bacillus  No.  1.  161 


Opsonins,  27 


Pails,  bacteria  in,  100 
Paracasein,  14 
Paratyphoid  group,  161 
Pasteurised  milk,  105 

cream  line  in,  185 

enzymes  in,  186 

Ottawa  results,  205 
Peptonising  organisms,  194 
Peroxidases,  23 

effect  of  heat,  188 

estimation  of,  91 

Physical  characteristics  of  milk,  28 
Plating  technique,  123,  125 
Ponder's  stain,  208 
Preservatives,  80 
Precipitins,  27 


220 


SUBJECT  INDEX 


Proteids,  6 
estimation  of,  73 
mucoid,  18 
whey,  14 

R 

Recknagel  phenomenon,  29 
Reductases,  24 

effect  of  heat  on,  188 

estimation  of,  89 
Refractive  index,  32,  79 

limits  for,  57 

Rennin,  effect  of  heat  on,  189 
Results,  calculation  of,  142 

recording,  206 

S 

Saccharate  of  lime,  87 
Salicylic  acid,  84 
Salolase,  22 
Salts,  19 

Samples,  collection  of,  202 
Schardinger's  reagent,  89 
Seasonal  variation  in  milk,  40 
Septic  sore  throat,  150 
Serum,  19,  57,  78 
Skim  milk,  88 
Solids-not-fat,  44 
Specific  gravity,  28 

determination  of,  69 
Specific  heat,  32 
Staphylococcus  pyogenes,  150 
Standards  for  milk,  59 

tables,  63 
Starch,  detection  of,  87 


Streptococci : 

biochemical  characteristics,  158 

faecal,  147 

haemolytic,  151 

pathogenic,  148,  153 
Streptococcus  lacticus,  109,  119,  152, 
153 

mastitidis,  150 

pyogenes,  152 
Stoppings,  50 

bacteria  in,  96 
Surface  tension,  32 


Toisson's  solution,  207 
Total  solids,  estimation  of,  69 

tables  for  calculating,  210-213 
Toxicity  of  milk,  114 

of  pasteurised  milk,  116 
Toxins,  27 

U 
Udder,  bacteria  in,  95 

influence  of  wiping,  washing,  etc., 


Viscogen,  87 
Viscosity,  60 
Voges  and  Proskauer  reaction,  136, 

145 
Volume  change  with  temperature, 

29,30 

Z 

Ziehl-Neelson  method  for  tubercle 
bacilli,  164 


NAME   INDEX 


A 


Aitkens,  30 
Alexander,  162 
Anderson,  167 
Andre  wes,  150 
Arthus,  28 
Ayers,  106,  194 


B 


Babcock,  22,  92,  181 

Backhaus,  97,  99,  100 

Bailey,  93 

Bang,  190 

Barthol,  130 

Batchelder,  94 

Bechamp,  17,  22 

Beger,  39 

Bellei,  91 

Benzynski,  55 

Berberich,  32 

Besredka,  28 

Block,  166 

Blyth,  21 

Borden,  121 

Boseley,  71,  81 

Bosworth,  7,  8,  11,  15,  20 

Boussingault,  50 

Bowhill,  156 

Breed,  128,  171,  178,  179 

Brew,  129 

Briot,  15 

Broadhurst,  155 

Browning,  160 

Buckley,  174 

Bunge,  34 


Burow,  8 
Burr,  32 


Cameron,  12 

Capps,  152 

Chamot,  140 

Chappelean,  198 

Chittenden,  8 

Clark,  121,  145 

Cook,  37 

Conn,  108,  117,  121,  124 

Corbett,  191 


Davis,  152 
Dean,  156 
Detepine,  115,  164,  166,  168,  180, 

181 

Desmouliers,  22 
Doane,  171,  174 
Dodd,  167 
Doll,  39 
Duclaux,  15 
Dugelli,  198 


Eastwood,  167,  168 
Eckles,  38,  42,  45,  50 
Ellenberger,  8 
Engling,  53 
Ernst,  171,  172 
Esten,  108 
Evans,  190 
Eyre,  157 


221 


222 


NAME  INDEX 


Fingerling,  39 
Fleishmann,  29,  32 
Fred,  130,  198 
Freudenreich  von,  22,  94 

G 

Geake,  8,  15 
Gerber,  181,  197 
Gillet,  22 
Glenn,  119 
Good,  191 
Gooderich,  127 
Griffiths,  167,  168 


Hall,  93 

Hammer,  111 

Hammerstein,  8,  14 

Hancke,  39 

Harden,  15 

Harrison,  98,  100 

Hastings,  111 

Hehner,  81 

Heidemann,  119,  152 

Heintz,  12 

Hempel,  8 

Henderson,  93,  95 

Hewarden,  16 

Hewlett,  18,  171,  177,  179 

Heyman,  196 

Hills,  37 

Hoffmann,  174,  178 

Holder,  150 

Holt,  162 

Houston,  181 

Hurst,  12 

J 

Jackoby,  17 
Jackson,  31,  152,  160 
Jensen,  22,  54,  130,  197 
Joannovico,  167 
Johnson,  106,  194 


K 

Kapsammer,  167 
Kastle,  22,  23 
Kaufman,  3 
Klein,  156,  158,  198 
Koning,  22,  35 
Koster,  14 
Krumwiede,  151 


Lacqueur,  8 
Lagne,  3 
Landtsheer,  22 
Lederle,  121 
Ledingham,  161 
Lehmann,  8 
Leonard,  81 
Levine,  145 
Lewis,  162 
Lindet,  18 
Liwschiz,  15 
Lobeck,  91 
Loevenhart,  15 
Loew,  23 
Lohnis,  198 
Long,  9,  10 
Lubs,  145 
Lythgoe,  32,  35,  86 

M 

Macallum,  15 
Malme'jac,  40 
Marfan,  22 
Marshall,  156 
Mathaiopoulos,  9 
McConkey,  94,  136 
McCrady,  142,  147 
McFadyean,  190 
Melia,  160 
Merklen,  22 
Michaelis,  171 
Miessner,  28 
Miller,  75,  130,  171 


NAME  INDEX 


223 


Monier- Williams,  83 
Morgan,  161 
Morgen,  39 
Morgenrath,  17 
Moro,  22 
Mule,  22 
Muller,  152 


Nobe*court,  22 
North,  121,  185 


N 


O 


O'Brien,  162 
Olsen,  50 

Orr,  98,  100,  137,  162 
Otto,  27 


Painter,  8 
Park,  94,  102,  162 
Pennington,  105,  111 
Peter,  91,  198 
Porch,  23 
Prescott,  178 

R 

Race,  194 

Rahe,  196 

Raudnitz,  17,  23 

Revenel,  111 

Revis,  171,  177,  181 

Richmond,  H.  D.,  6,  8,  29,  30,  37, 

44,  60,  71,  75,  81,  87 
Richmond,  S.  O.,  29 
Robertson,  9 
Rogers,  137 
Romer,  89 
Rosam,  128 
Rosenau,  M.  J.,  102 
Ross,  162 
Rothera,  31 
Rothenfusser,  91 


Rueduger,  154 

Rullman,  23 

Rupp,  189 

Russell,  22,  92,  174,  178 


Sackur,  8 
Salge,  196 
Savage,  101,  143,  147,  150, 158, 171, 

176,  178,  179 
Schaffer,  55 
Schardinger,  89 
Schern,  89 
Schmidt,  14 
Schnorf ,  54 
Scholberg,  162 
Schrewsbury,  82 
Schroeder,  182 
Schroeter,  198 
Schryver,  7,  15 
Sebelein,  18 
Sedgwick,  94 
Seligman,  22 
Shaw,  38,  42,  45,  50 
Sherwood,  140 
Sieglin,  39 
Skar,  128 
Slack,  126,  174 

Slyke,  L.  L.  Van,  7,  8,  11,  15,  20 
Slyke,  D.  D.  Van,  10 
Smith,  Graham,  162 
Soldner,  8 
Sothurst,  53 
Spolverini,  22 
Sprague,  178 
Stewart,  126,  174 
St.  John,  105 
Stidger,  179 
Stribald,  190 
Stocking,  96,  97,  99,  105 
Stockman,  190 
Stohman,  2 
Stokes,  87,  171 


224 


NAME  INDEX 


Stone,  178 
Storch,  18 
Strewe,  19 


Tange,  8 
Taylor,  30 
Thoni,  94 
Thomson,  83 
Thornton,  160 
Timpe,  45 
Todd,  156 
Tonney,  160,  161,  182 


Valentine,  151 
Velde  der,  22 


Vieth,  37 
Villar,  171,  177 

Wallis,  162 
Walter,  197 
Ward,  93,  95 
Wegefarth,  171 
Weigner,  29,  30 
Wender,  22 
Wilkinson,  91 
Willem,  22 
Winkler,  171 
Winslow,  155 

Zaitschik,  22 
Zielstorff,  39 


W 


RB  17-60m-6>'59 
(A2840slO)4l88 


iYB  47243 


415024 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


